Highly sensitive microplate ß-galactosidase assay for yeast two-hybrid systems

Brouchon-Macari, L., Joseph, M., Dagher, M. Highly sensitive microplate ß-galactosidase assay for yeast two-hybrid systems. BioTechniques Vol. 35, No. 3, 446-448 (2003).

“Culture for 20 h with shaking in a deep-well, conical-bottom microplate.” (Yeast Culture protocol)

“Shake at 800 rpm for 10 min at 30°C.” (ß-Galactosidase Assay)

Localized transfection on arrays of magnetic beads coated with PCR products

salan, M., Santori, M., Gonzalez, C., Serrano, L. Localized transfection on arrays of magnetic beads coated with PCR products. Nature MethodsVol. 2, No. 2,113-118 (2005).

“We pipetted 10 µl bead aliquots in a 96-well plate, adding 5 µl of water plus 5 µl of PCR product per well. After mixtures were incubated for 3 h at 22 °C, with mixing at 800 r.p.m, ...”

“We incubated the plate for 45 min at 22 °C with mixing at 800 r.p.m ...”

Microplate Nitrate Test Kit — Standard Range

Microplate Nitrate Test Kit — Standard Range, The Nitrate Elimination Co., Inc., Lake Linden, Michigan.

“Pipet 90 µl NaR-Assay Buffer-NADH solution (prepared in Step 8 of Reagent Preparation) to each well. Shake on a plate mixer for ~20 minutes at 800 rpm.”

“Add 50 µl Color Reagent No. 1 and 50 µl Color Reagent No. 2 solution to each well. Shake on a plate mixer for ~10 minutes at 800 rpm.”

Scalable high-throughput micro-expression device for recombinant proteins

Page, R. et al. Scalable high-throughput micro-expression device for recombinant proteins. BioTechniques Vol. 37, No. 3, 364-370 (2004).

“[sterile 2-mL, 96-deep-well, round-bottom blocks are] grown at 900 rpm and 37°C.”

“...cultures are allowed to shake at 900 rpm and 37°C for an additional 4-5 h.”

Culture of Human Cervical Epithelial Cells

Stanley, M., 2002, ‘Culture of Human Cervical Epithelial Cells’, Culture of Epithelial Cells, Second Edition, Freshney, R., Freshney, M. (eds.), Wiley-Liss, Inc.: New York pp. 137-169.

“Place the universal on a magnetic stirrer in an incubator or hot room at 37°C and stir slowly for 30-40 min.”

Determination of Hydrogen Peroxide (H2O2) Residual in Fiber Matrices

Determination of Hydrogen Peroxide (H2O2) Residual in Fiber Matrices, Solvay Chemicals, Inc., 2004, Houston, Texas.

“2. Add 50mL of water and a small magnetic stir bar to the flask containing the sample. 3. Agitate for 15 minutes using a magnetic stirring device. Make sure the slurry is agitated sufficiently to stay suspended during this period. 4. Turn off the stirrer. 5. Add 20 mL of... Swirl to mix. 6. Add 25 ml of... Swirl to mix. 7. Let the slurry stand for 5 minutes... 8. Resume stirring.”

“12. Turn off the stirrer and let the slurry stand for 2.0 hours... 13. Resume stirring and titrate as in “11.” above.

Method 9213: Potentiometric Determination Of Cyanide In Aqueous Samples and Distillates With Ion-Selective Electrode

United States Environmental Protection Agency. “Method 9213: Potentiometric Determination Of Cyanide In Aqueous Samples And Distillates With Ion-Selective Electrode” EPA Publication SW-846: Test Methods For Evaluating Solid Waste, Physical/Chemical Methods. Washington: GPO, 1996.

“Add a PTFE-coated magnetic stir bar, place the beaker on a magnetic stir plate, and stir at slow speed (no visible vortex).

Preparation and characterisation of chitosan and trimethyl-chitosan modified PCL nanoparticles as DNA carriers

Haas, J. et al. Preparation and characterisation of chitosan and trimethyl-chitosan modified PCL nanoparticles as DNA carriers. AAPS PharmSciTech. 2004.

“As for the aqueous phase, 100 mg PVA and 15 mg CS or TMC were stirred in 5 ml water for 2 hours at room temperature until a clear solution was obtained. The organic phase was passed through a 0.22µm syringe filter to remove any un-dissolved solids and subsequently added drop-wise to the aqueous phase under constant stirring. The resulting micro-emulsion was kept under constant agitation on magnetic stirrer at 1,000 rpm for 1 hour...”

“This colloidal preparation was diluted to a volume of 50 ml by adding water drop wise under stirring conditions (1,000 rpm, magnetic stirrer), which resulted in nanoprecipitation.”

Synthesis of (-)-(E,S)-3-(Benzyloxy)-1-Butenyl Phenyl Sulfone via a Horner-Wadsworth-Emmons Reaction of (-)-(S)-2-(Benzyloxy) Propanal

‘Synthesis of (-)-(E,S)-3-(Benzyloxy)-1-Butenyl Phenyl Sulfone via a Horner-Wadsworth-Emmons Reaction of (-)-(S)-2-(Benzyloxy) Propanal’, Organic Syntheses, Collective Volume 10, Freeman, J. (ed.), Wiley: New York, p. 66; Vol. 78, p. 177, 2004.

“The reaction mixture is stirred for 48 to 60 hr at room temperature (Note 7) and subsequently diluted with water (100mL) and hexane (300 mL). Stirring is continued for an additional 3 hr at room temperature.”

“...is added very slowly dropwise to the solution of the ester and stirring is continued for at least 1 hr after the complete addition of the DIBAH solution (Note12). Upon complete consumption of the ester, the crude reaction mixture is poured directly with vigorous stirring into 360 mL of...”

“The aldehyde solution is added dropwise at 0°C with vigorous stirring. After complete addition the reaction mixture is stirred for ca. 12 hr and allowed to warm...”

Culture of Human Cervical Epithelial Cells

Stanley, M., 2002, ‘Culture of Human Cervical Epithelial Cells’, Culture of Epithelial Cells, Second Edition, Freshney, R., Freshney, M. (eds.), Wiley-Liss, Inc.: New York pp. 137-169.

“Place the universal on a magnetic stirrer in an incubator or hot room at 37°C and stir slowly for 30-40 min.”

Determination of Hydrogen Peroxide (H2O2) Residual in Fiber Matrices

Determination of Hydrogen Peroxide (H2O2) Residual in Fiber Matrices, Solvay Chemicals, Inc., 2004, Houston, Texas.

“2. Add 50mL of water and a small magnetic stir bar to the flask containing the sample. 3. Agitate for 15 minutes using a magnetic stirring device. Make sure the slurry is agitated sufficiently to stay suspended during this period. 4. Turn off the stirrer. 5. Add 20 mL of... Swirl to mix. 6. Add 25 ml of... Swirl to mix. 7. Let the slurry stand for 5 minutes... 8. Resume stirring.”

“12. Turn off the stirrer and let the slurry stand for 2.0 hours... 13. Resume stirring and titrate as in “11.” above.

Method 9213: Potentiometric Determination Of Cyanide In Aqueous Samples and Distillates With Ion-Selective Electrode

United States Environmental Protection Agency. “Method 9213: Potentiometric Determination Of Cyanide In Aqueous Samples And Distillates With Ion-Selective Electrode” EPA Publication SW-846: Test Methods For Evaluating Solid Waste, Physical/Chemical Methods. Washington: GPO, 1996.

“Add a PTFE-coated magnetic stir bar, place the beaker on a magnetic stir plate, and stir at slow speed (no visible vortex).

Preparation and characterisation of chitosan and trimethyl-chitosan modified PCL nanoparticles as DNA carriers

Haas, J. et al. Preparation and characterisation of chitosan and trimethyl-chitosan modified PCL nanoparticles as DNA carriers. AAPS PharmSciTech. 2004.

“As for the aqueous phase, 100 mg PVA and 15 mg CS or TMC were stirred in 5 ml water for 2 hours at room temperature until a clear solution was obtained. The organic phase was passed through a 0.22µm syringe filter to remove any un-dissolved solids and subsequently added drop-wise to the aqueous phase under constant stirring. The resulting micro-emulsion was kept under constant agitation on magnetic stirrer at 1,000 rpm for 1 hour...”

“This colloidal preparation was diluted to a volume of 50 ml by adding water drop wise under stirring conditions (1,000 rpm, magnetic stirrer), which resulted in nanoprecipitation.”

Synthesis of (-)-(E,S)-3-(Benzyloxy)-1-Butenyl Phenyl Sulfone via a Horner-Wadsworth-Emmons Reaction of (-)-(S)-2-(Benzyloxy) Propanal

‘Synthesis of (-)-(E,S)-3-(Benzyloxy)-1-Butenyl Phenyl Sulfone via a Horner-Wadsworth-Emmons Reaction of (-)-(S)-2-(Benzyloxy) Propanal’, Organic Syntheses, Collective Volume 10, Freeman, J. (ed.), Wiley: New York, p. 66; Vol. 78, p. 177, 2004.

“The reaction mixture is stirred for 48 to 60 hr at room temperature (Note 7) and subsequently diluted with water (100mL) and hexane (300 mL). Stirring is continued for an additional 3 hr at room temperature.”

“...is added very slowly dropwise to the solution of the ester and stirring is continued for at least 1 hr after the complete addition of the DIBAH solution (Note12). Upon complete consumption of the ester, the crude reaction mixture is poured directly with vigorous stirring into 360 mL of...”

“The aldehyde solution is added dropwise at 0°C with vigorous stirring. After complete addition the reaction mixture is stirred for ca. 12 hr and allowed to warm...”

Calcium/Calmodulin-dependent Protein Kinase Activates Serum Response Factor Transcription Activity by Its Dissociation from Histone Deacetylase, HDAC4

Davis, F., Gupta, M., Camoretti-Mercado, B., Schwartz, R., and Gupta, M. Calcium/Calmodulin-dependent Protein Kinase Activates Serum Response Factor Transcription Activity by Its Dissociation from Histone Deacetylase, HDAC4. The Journal of Biological Chemistry Vol. 278, Issue 22, 20047-20058, (2003).

“Protein A/G-agarose (20µl) was then added to each sample and incubated at 4°C overnight on a rotating device.”

”...reactions were continued at room temperature with continuous rocking for an additional 1 h.”

Genomic Analysis of Stationary-Phase and Exit in Saccharomyces cerevisiae: Gene Expression and Identification of Novel Essential Genes Martinez, M., et al. Genomic Analysis of Stationary-Phase and Exit in Saccharomyces cere

Martinez, M., et al. Genomic Analysis of Stationary-Phase and Exit in Saccharomyces cerevisiae: Gene Expression and Identification of Novel Essential Genes. Molecular Biology of the Cell Vol. 15, 5295-5305 (2004).

“Before microarray printing, ...were incubated at 21°C in 48-52% humidity for 4-20 h...”

”Slides were incubated in single-slide capacity chambers...on a rotating platform at 42°C for 16-20 h.“

Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation

Boutahar, N., Guignandon, A., Vico, L., and Lafage-Proust, M. Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation. The Journal of Biological Chemistry Vol. 279, Issue 29, 30588-30599, (2004).

“After incubation at 4 °C for 2 h on a rotating device, immune complexes were precipitated at 4 °C overnight on a rotating device by using protein-A Sepharose...”

“After transfer, membranes were blocked using 5% nonfat dried milk in PBS, pH 7.4, and incubated overnight at 4 °C with the...”

Role of bone marrow cell trafficking in replenishing skeletal muscle SP and MP cell populations

Rivier, F. et al. Role of bone marrow cell trafficking in replenishing skeletal muscle SP and MP cell populations. Journal of Cell Science Vol. 117, 1979-1988 (2004).

“Muscles were finely minced using sterile scalpels and digested at 37°C in 1U ml-1 dispase (Roche) and 2 mg ml-1 collagenase-type 4 (Worthington) for 45 minutes using an Enviro-Genie...”

“100 ng of denatured probe were added to each slide and sections were hybridized overnight at 37°C in a humidified chamber.”

“Slides were incubated in a humidified chamber at 37°C for 30 minutes with digoxigenin blocking solution... then for an additional 30 minutes with...”

Screening lambda cDNA or genomic libraries

Koelle, M. 1997, “Screening lambda cDNA or genomic libraries”, Yale University. Available from: http://info.med.yale.edu/mbb/koelle/protocols/protocol_lambda_library_sc.html.

“Add the probe to the blot/hyb. Solution, mix well, and incubate overnight at 65° on a rocker...”

“Place on a rotating platform at room temp for 1-2 hours.”

Structure — function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis

Jennifer A. Beckstead, Michael N. Oda, Dale D. O. Martin, Trudy M. Forte, John K. Bielicki, Trish Berger, Robert Luty, Cyril M. Kay, and Robert O. Ryan. Structure — function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis” Biochemistry, 42 (31), pp 9416 - 9423 (2003).

“Subsequently, the IgG fraction was biotinylated by incubation for 15 min with 5 ml 0.5 mg/ml sulfo-NHS-Ssbiotin... in borate buffer on a rocking platform with slow agitation.”

“Non specific binding sites on the membrane were blocked with 0.1% TTBS...overnight, at room temperature while rotating.”

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts

Hirozane-Kishikawa, T. et al. Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts. BioTechniques Vol. 35, No. 3, 510-518 (2003).

“One microliter of 5 M NaCl was then added to the driver/tester mixture, and the hybridization was performed at 4°C for 8 h...”

“The hybridized sample library was diluted with 500 µL HAP buffer, incubated at 65°C, and added to the HAP, mixed vigorously, and rotated at 60°C for 30 min.”

“The HAP was washed twice with 500 µL HAP buffer, rotating at 60°C for 30 min...”

Calcium/Calmodulin-dependent Protein Kinase Activates Serum Response Factor Transcription Activity by Its Dissociation from Histone Deacetylase, HDAC4

Davis, F., Gupta, M., Camoretti-Mercado, B., Schwartz, R., and Gupta, M. Calcium/Calmodulin-dependent Protein Kinase Activates Serum Response Factor Transcription Activity by Its Dissociation from Histone Deacetylase, HDAC4. The Journal of Biological Chemistry Vol. 278, Issue 22, 20047-20058, (2003).

“Protein A/G-agarose (20µl) was then added to each sample and incubated at 4°C overnight on a rotating device.”

”...reactions were continued at room temperature with continuous rocking for an additional 1 h.”

Genomic Analysis of Stationary-Phase and Exit in Saccharomyces cerevisiae: Gene Expression and Identification of Novel Essential Genes Martinez, M., et al. Genomic Analysis of Stationary-Phase and Exit in Saccharomyces cere

Martinez, M., et al. Genomic Analysis of Stationary-Phase and Exit in Saccharomyces cerevisiae: Gene Expression and Identification of Novel Essential Genes. Molecular Biology of the Cell Vol. 15, 5295-5305 (2004).

“Before microarray printing, ...were incubated at 21°C in 48-52% humidity for 4-20 h...”

”Slides were incubated in single-slide capacity chambers...on a rotating platform at 42°C for 16-20 h.“

Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation

Boutahar, N., Guignandon, A., Vico, L., and Lafage-Proust, M. Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation. The Journal of Biological Chemistry Vol. 279, Issue 29, 30588-30599, (2004).

“After incubation at 4 °C for 2 h on a rotating device, immune complexes were precipitated at 4 °C overnight on a rotating device by using protein-A Sepharose...”

“After transfer, membranes were blocked using 5% nonfat dried milk in PBS, pH 7.4, and incubated overnight at 4 °C with the...”

Role of bone marrow cell trafficking in replenishing skeletal muscle SP and MP cell populations

Rivier, F. et al. Role of bone marrow cell trafficking in replenishing skeletal muscle SP and MP cell populations. Journal of Cell Science Vol. 117, 1979-1988 (2004).

“Muscles were finely minced using sterile scalpels and digested at 37°C in 1U ml-1 dispase (Roche) and 2 mg ml-1 collagenase-type 4 (Worthington) for 45 minutes using an Enviro-Genie...”

“100 ng of denatured probe were added to each slide and sections were hybridized overnight at 37°C in a humidified chamber.”

“Slides were incubated in a humidified chamber at 37°C for 30 minutes with digoxigenin blocking solution... then for an additional 30 minutes with...”

Screening lambda cDNA or genomic libraries

Koelle, M. 1997, “Screening lambda cDNA or genomic libraries”, Yale University. Available from: http://info.med.yale.edu/mbb/koelle/protocols/protocol_lambda_library_sc.html.

“Add the probe to the blot/hyb. Solution, mix well, and incubate overnight at 65° on a rocker...”

“Place on a rotating platform at room temp for 1-2 hours.”

Structure — function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis

Jennifer A. Beckstead, Michael N. Oda, Dale D. O. Martin, Trudy M. Forte, John K. Bielicki, Trish Berger, Robert Luty, Cyril M. Kay, and Robert O. Ryan. Structure — function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis” Biochemistry, 42 (31), pp 9416 - 9423 (2003).

“Subsequently, the IgG fraction was biotinylated by incubation for 15 min with 5 ml 0.5 mg/ml sulfo-NHS-Ssbiotin... in borate buffer on a rocking platform with slow agitation.”

“Non specific binding sites on the membrane were blocked with 0.1% TTBS...overnight, at room temperature while rotating.”

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts

Hirozane-Kishikawa, T. et al. Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts. BioTechniques Vol. 35, No. 3, 510-518 (2003).

“One microliter of 5 M NaCl was then added to the driver/tester mixture, and the hybridization was performed at 4°C for 8 h...”

“The hybridized sample library was diluted with 500 µL HAP buffer, incubated at 65°C, and added to the HAP, mixed vigorously, and rotated at 60°C for 30 min.”

“The HAP was washed twice with 500 µL HAP buffer, rotating at 60°C for 30 min...”

Eotaxin Represents the Principal Eosinophil Chemoattractant in a Novel Murine Asthma Model Induced by House Dust Containing Cockroach Allergens

Kim, J. et al. Eotaxin Represents the Principal Eosinophil Chemoattractant in a Novel Murine Asthma Model Induced by House Dust Containing Cockroach Allergens. The American Association of Immunologists Vol. 167, No. 5, 2808-2815 (2001).

“Sterile PBS (2 ml) was added to the dust, which was then mixed end-over-end on an orbital rotator (Roto-Shake Genie; Scientific Industries, Bohemia, NY) at 4°C for 16 h”

How VASP enhances actin-based motility

Samarin, S. et al. How VASP enhances actin-based motility. The Journal of Cell Biology Vol. 163, No. 1, 131-142 (2003).

“...were incubated with ActA at the indicated concentrations for 1 h at 4°C on a Roto-Shake machine, and processed...”

Roto-Shake Genie Personal Communication #1

Setser, E., personal communication, 2003.

“We have recently purchased the Roto-Shake Genie and, while it satisfies our rocking or rotating needs, it also provides another benefit to our lab. We are able to use the flat, magnetic surface to pellet our Dynabeads Oligo (dT) 25 during the purification of mRNA. Good quality mRNA is essential to many of our procedures and the use of magnetic beads makes purification very speedy. Using the Roto-Shakes magnet for this procedure simply requires placing the sample tubes on the surface of the platform for pelleting. There is no need to purchase another magnet to do the isolation and means one less item setting around on the bench top.”

Roto-Shake Genie Personal Communication #2

Tan, J., personal communication, 2003.

“The Roto-Shake Genie attains the standard expectations of a rocker: the beveled non-skid tray provides guard-rails for evenly shaking blots, and adjustable speed permits experiment-appropriate washing or mixing force. However, the Roto-Shake Genie provides additional design features which set it apart from the standard rotator/shaker. Choice from five different rotating or mixing motions in one apparatus renders this model applicable to a broad range of needs. Beyond traditional applications of blot incubation or solution mixing, the Genie is fit for virus and protein-protein interaction, or protein-nucleic acid interaction, techniques. Examples include the popular Baculovirus system for recombinant protein production; the Roto-Shake Genie combined rotating/rocking setting offers even distribution of recombinant virus over insect cells during the prescribed infection incubation. For experiments demanding tube rotation, including any variety of affinity matrix incubation with protein or nucleic acid target, the magnetic adaptors provided with this purchase firmly secure samples for extended mixing. Magnetic holders are also included to fasten seal-a-meal bags as used for radioactive probe mixing as mandated in Southerns, Northerns, or nuclear run-ons. Given Roto-Shake Genie adaptability to a temperature range of 0 to 38°C, moving the Genie to a cold room or warm incubator setting offers an alternative to the potential clean-up mess of radioactive blot incubation in a waterbath shaker.”

“...we are eager to use the Roto-Shake Genie for co-immunoprecipitation of proteins, for which the magnetic tubeholders, and option to move the Genie from a refrigerated setting to a countertop, room temperature setting, are particularly helpful. The magnetic tubeholders should preclude tubes sliding off the shaker mid-incubation, as occurred with our older shaker, and the temperature flexibility permits antibody vs. affinity matrix binding at distinct temperatures. Our lab also uses the Baculovirus system as mentioned above, which requires shaking incubation of virus and target cells. My own project has called for nuclear run-on analysis, and a deluge of Southerns, for which this rocker would certainly be helpful. Finally, Western blotting is an almost constant activity in our lab and the Genie is well-suited to serve in that capacity.”

Structural, functional and comparative studies of human chromosome 22q13.31

Goward, M., 2002, “Structural, functional and comparative studies of human chromosome 22q13.31”, PhD thesis, Cambridge University.

“The filters were then left to pre-hybridise at 65°C for at least 3 hours, in an orbital shaker.”

“The filters were then rinsed twice in 2 X SSC at room temperature, followed by two 30 minute washes with 0.5 X SSC/1% N-Lauroyl Sarcosine at 65°C in an orbital shaker.”

“Following electrophoresis, the gels were trimmed to 19 cm, placed in plastic trays containing Vistra Green stain mix and agitated in the dark, on an orbital platform shaker, for 45 minutes.”

Analysis of Proteins by Immunoprecipitation

P.J. Hansen, Department of Animal Sciences, University of Florida.

“ ...(shake to suspend slurry before pipetting) and incubate on a tube turner for 6 h at room termperature.

Protocol for Reconstituting Tissue Factor (TF) into Phospholipid Vesicles using Bio-Beads SM-2

Smilth SA & Morrissey JH, (2004) Rapid and efficient incorporation of tissue factor into liposomes. J. Thromb. Haemost. 2: 1155-1162.

“Rotate the bottle on the tube rotator for 15 min at about 20 rpm.”

“Rotate for 90 min on tube rotator at about 20 rpm.”

Protocol for Isolation of Vicinal Cis-Diol Containing Sugar Compounds with SiMAG-Boronic Acid

chemicell GmbH, www.chemicell.com.

“Incubate at room temperature for 15-30 minutes by gentle mixing (tube rotator).”

Basic Protocol: Immunoisolation of GLUT4 Vesicles from Rat Adipose Cells

Cp Current Protocols, www.currentprotocols.com.

“Tube rotator ...”

Analysis of Proteins by Immunoprecipitation

P.J. Hansen, Department of Animal Sciences, University of Florida.

“ ...(shake to suspend slurry before pipetting) and incubate on a tube turner for 6 h at room termperature.

Protocol for Reconstituting Tissue Factor (TF) into Phospholipid Vesicles using Bio-Beads SM-2

Smilth SA & Morrissey JH, (2004) Rapid and efficient incorporation of tissue factor into liposomes. J. Thromb. Haemost. 2: 1155-1162.

“Rotate the bottle on the tube rotator for 15 min at about 20 rpm.”

“Rotate for 90 min on tube rotator at about 20 rpm.”

Protocol for Isolation of Vicinal Cis-Diol Containing Sugar Compounds with SiMAG-Boronic Acid

chemicell GmbH, www.chemicell.com.

“Incubate at room temperature for 15-30 minutes by gentle mixing (tube rotator).”

Basic Protocol: Immunoisolation of GLUT4 Vesicles from Rat Adipose Cells

Cp Current Protocols, www.currentprotocols.com.

“Tube rotator ...”

Cell Disruption Using Glass Bead Media

Spherical lead free soda lime glass beads are commonly used for mechanical disruption of many yeast, bacterial and soil samples. Glass beads of a pre-determined size and volume are placed in a 1.5ml or 2.0ml microtube along with a pre-determined sample amount. The closed tube is then shaken vigorously at high speed, causing collisions between the glass beads and sample material. Scientific Industries’ Disruptor Genie® and TurboMix™ attachment for the Vortex-Genie 2 Family of Mixers are excellent choices for this process as they both simultaneously agitate and vortex at high speed, dramatically increasing cell or sample disruption. Each can hold up to twelve 1.5 ml or 2.0 ml microtubes at once. The disrupted cells may be removed after shaking for downstream processing.

Scientific Industries’ Disruptor Beads are packaged as 375g (8 fl. oz.) bottles in two sizes:

• 0.1 mm diameter beads (Catalog No. SI-BG01) — For use with Bacteria
• 0.5 mm diameter beads (Catalog No. SI-BG05) — For use with Yeast/Fungi

Care And Cleaning

Pre-preparation steps for Scientific Industries’ Disruptor Beads are generally unnecessary. If desired, they may be soaked in a 1:8 dilution of household bleach for 20 minutes, rinsed with copious amounts of distilled or RO water, and baked at 50 to 65°C for a minimum of 2 hours, or until completely dry. If the glass beads do not pour freely, repeat the cleaning and drying process. Disruptor Beads may also be autoclaved after proper disinfecting or cleaning.

The Disruptor Beads may be reused, if desired, after proper disinfecting or cleaning and autoclaving. Subsequent uses and excessive handling of the beads may result in the creation of fines, which could adversely affect cell disruption efficiency. As such, it is not recommended to frequently reuse Disruptor Beads.

Disruptor Beads may be stored at room temperature or frozen in an airtight container prior to use. In addition, the Disruptor Genie and TurboMix attachment for the Vortex-Genie 2 Family of Mixers may be used in cold rooms.

Sample Application Methods

NOTE: DETAILED DIRECTIONS FOR USE WILL DIFFER DEPENDING ON THE INDIVIDUAL PROTOCOL USED OR THE OUTCOME DESIRED. THE SAMPLE METHODS BELOW ARE EXAMPLES ONLY.

Bacteria Disruption:
Disruptor Beads, 0.1 mm diameter, are recommended for disruption of bacterial samples. A typical sample ratio would be 50% Disruptor Beads to 50% bacterial suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and bacterial suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Disruption at room temperature using chilled materials for 3 to 5 minutes at highest speed should be sufficient to recover 85% of the bacterial RNA. Disruption can be performed in a cold room as well. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Protocol for protein expression in Escherichia coli from a T7 expression system:
The following protocol is designed to provide approximately 1.5 mls of lysed cell supernatant that can be used for subsequent analyses. Inoculate 2 ml of Luria Broth (plus antibiotic) with an appropriate E. coli strain (i.e. BL21 DE3) containing an expression plasmid encoding the protein of interest. Incubate the culture at 37°C with shaking overnight. Inoculate the 2ml overnight culture into 40 mls of LB (plus antibiotic) and incubate until mid-log phase of growth (A600 = 0.4 - 0. 6). This step normally takes less than 2 hours. Add IPTG to 0.5 mM and incubate the culture for an additional 4 hours or more. Harvest the cells by centrifugation and then resuspend the cell pellet with 1.8 mls of buffer (50 mM TrisHCl, pH 7.5 or any other suitable buffer). Transfer 0.6 mls of the cell suspension to a 2 ml microtube and add 0.2 gms of 0.1 mm disruptor beads. Using greater quantities of beads (up to 0.5 gms) did not increase the efficiency of cell lysis. Close the tube and shake vigorously for 2 minutes with the TurboMix attachment to the Vortex-Genie or Disruptor Genie. Pellet the cells by centrifugation at maximum speed for 5 minutes in a microfuge. Take an aliquot of the supernatant for SDS-PAGE analysis and decant the rest of the supernatant into a new tube.

Yeast/Fungi Disruption:
Disruptor Beads, 0.5 mm diameter, are recommended for disruption of yeast or fungi samples. A typical sample ratio would be 50% Disruptor Beads to 50% of yeast or fungus suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and yeast or fungus suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Yeast cells and fungi are generally more difficult to shear than bacterial cells, so increased disruption times may be necessary. Disruption in a cold room with chilled materials for 5 to 7 minutes at highest speed should be sufficient to disrupt the cell sample. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Soil Sample Disruption:
Either size of Disruptor Beads can be used for soil samples. A typical sample ratio would be 50% Disruptor Beads to 50% soil sample suspension by volume. Allow head space (~20%) within the microtube to facilitate disruption action. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Cell Disruption Using Glass Bead Media

Spherical lead free soda lime glass beads are commonly used for mechanical disruption of many yeast, bacterial and soil samples. Glass beads of a pre-determined size and volume are placed in a 1.5ml or 2.0ml microtube along with a pre-determined sample amount. The closed tube is then shaken vigorously at high speed, causing collisions between the glass beads and sample material. Scientific Industries’ Disruptor Genie® and TurboMix™ attachment for the Vortex-Genie 2 Family of Mixers are excellent choices for this process as they both simultaneously agitate and vortex at high speed, dramatically increasing cell or sample disruption. Each can hold up to twelve 1.5 ml or 2.0 ml microtubes at once. The disrupted cells may be removed after shaking for downstream processing.

Scientific Industries’ Disruptor Beads are packaged as 375g (8 fl. oz.) bottles in two sizes:

• 0.1 mm diameter beads (Catalog No. SI-BG01) — For use with Bacteria
• 0.5 mm diameter beads (Catalog No. SI-BG05) — For use with Yeast/Fungi

Care And Cleaning

Pre-preparation steps for Scientific Industries’ Disruptor Beads are generally unnecessary. If desired, they may be soaked in a 1:8 dilution of household bleach for 20 minutes, rinsed with copious amounts of distilled or RO water, and baked at 50 to 65°C for a minimum of 2 hours, or until completely dry. If the glass beads do not pour freely, repeat the cleaning and drying process. Disruptor Beads may also be autoclaved after proper disinfecting or cleaning.

The Disruptor Beads may be reused, if desired, after proper disinfecting or cleaning and autoclaving. Subsequent uses and excessive handling of the beads may result in the creation of fines, which could adversely affect cell disruption efficiency. As such, it is not recommended to frequently reuse Disruptor Beads.

Disruptor Beads may be stored at room temperature or frozen in an airtight container prior to use. In addition, the Disruptor Genie and TurboMix attachment for the Vortex-Genie 2 Family of Mixers may be used in cold rooms.

Sample Application Methods

NOTE: DETAILED DIRECTIONS FOR USE WILL DIFFER DEPENDING ON THE INDIVIDUAL PROTOCOL USED OR THE OUTCOME DESIRED. THE SAMPLE METHODS BELOW ARE EXAMPLES ONLY.

Bacteria Disruption:
Disruptor Beads, 0.1 mm diameter, are recommended for disruption of bacterial samples. A typical sample ratio would be 50% Disruptor Beads to 50% bacterial suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and bacterial suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Disruption at room temperature using chilled materials for 3 to 5 minutes at highest speed should be sufficient to recover 85% of the bacterial RNA. Disruption can be performed in a cold room as well. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Protocol for protein expression in Escherichia coli from a T7 expression system:
The following protocol is designed to provide approximately 1.5 mls of lysed cell supernatant that can be used for subsequent analyses. Inoculate 2 ml of Luria Broth (plus antibiotic) with an appropriate E. coli strain (i.e. BL21 DE3) containing an expression plasmid encoding the protein of interest. Incubate the culture at 37°C with shaking overnight. Inoculate the 2ml overnight culture into 40 mls of LB (plus antibiotic) and incubate until mid-log phase of growth (A600 = 0.4 - 0. 6). This step normally takes less than 2 hours. Add IPTG to 0.5 mM and incubate the culture for an additional 4 hours or more. Harvest the cells by centrifugation and then resuspend the cell pellet with 1.8 mls of buffer (50 mM TrisHCl, pH 7.5 or any other suitable buffer). Transfer 0.6 mls of the cell suspension to a 2 ml microtube and add 0.2 gms of 0.1 mm disruptor beads. Using greater quantities of beads (up to 0.5 gms) did not increase the efficiency of cell lysis. Close the tube and shake vigorously for 2 minutes with the TurboMix attachment to the Vortex-Genie or Disruptor Genie. Pellet the cells by centrifugation at maximum speed for 5 minutes in a microfuge. Take an aliquot of the supernatant for SDS-PAGE analysis and decant the rest of the supernatant into a new tube.

Yeast/Fungi Disruption:
Disruptor Beads, 0.5 mm diameter, are recommended for disruption of yeast or fungi samples. A typical sample ratio would be 50% Disruptor Beads to 50% of yeast or fungus suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and yeast or fungus suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Yeast cells and fungi are generally more difficult to shear than bacterial cells, so increased disruption times may be necessary. Disruption in a cold room with chilled materials for 5 to 7 minutes at highest speed should be sufficient to disrupt the cell sample. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Soil Sample Disruption:
Either size of Disruptor Beads can be used for soil samples. A typical sample ratio would be 50% Disruptor Beads to 50% soil sample suspension by volume. Allow head space (~20%) within the microtube to facilitate disruption action. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Cell Disruption Using Glass Bead Media

Spherical lead free soda lime glass beads are commonly used for mechanical disruption of many yeast, bacterial and soil samples. Glass beads of a pre-determined size and volume are placed in a 1.5ml or 2.0ml microtube along with a pre-determined sample amount. The closed tube is then shaken vigorously at high speed, causing collisions between the glass beads and sample material. Scientific Industries’ Disruptor Genie® and TurboMix™ attachment for the Vortex-Genie 2 Family of Mixers are excellent choices for this process as they both simultaneously agitate and vortex at high speed, dramatically increasing cell or sample disruption. Each can hold up to twelve 1.5 ml or 2.0 ml microtubes at once. The disrupted cells may be removed after shaking for downstream processing.

Scientific Industries’ Disruptor Beads are packaged as 375g (8 fl. oz.) bottles in two sizes:

• 0.1 mm diameter beads (Catalog No. SI-BG01) — For use with Bacteria
• 0.5 mm diameter beads (Catalog No. SI-BG05) — For use with Yeast/Fungi

Care And Cleaning

Pre-preparation steps for Scientific Industries’ Disruptor Beads are generally unnecessary. If desired, they may be soaked in a 1:8 dilution of household bleach for 20 minutes, rinsed with copious amounts of distilled or RO water, and baked at 50 to 65°C for a minimum of 2 hours, or until completely dry. If the glass beads do not pour freely, repeat the cleaning and drying process. Disruptor Beads may also be autoclaved after proper disinfecting or cleaning.

The Disruptor Beads may be reused, if desired, after proper disinfecting or cleaning and autoclaving. Subsequent uses and excessive handling of the beads may result in the creation of fines, which could adversely affect cell disruption efficiency. As such, it is not recommended to frequently reuse Disruptor Beads.

Disruptor Beads may be stored at room temperature or frozen in an airtight container prior to use. In addition, the Disruptor Genie and TurboMix attachment for the Vortex-Genie 2 Family of Mixers may be used in cold rooms.

Sample Application Methods

NOTE: DETAILED DIRECTIONS FOR USE WILL DIFFER DEPENDING ON THE INDIVIDUAL PROTOCOL USED OR THE OUTCOME DESIRED. THE SAMPLE METHODS BELOW ARE EXAMPLES ONLY.

Bacteria Disruption:
Disruptor Beads, 0.1 mm diameter, are recommended for disruption of bacterial samples. A typical sample ratio would be 50% Disruptor Beads to 50% bacterial suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and bacterial suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Disruption at room temperature using chilled materials for 3 to 5 minutes at highest speed should be sufficient to recover 85% of the bacterial RNA. Disruption can be performed in a cold room as well. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Protocol for protein expression in Escherichia coli from a T7 expression system:
The following protocol is designed to provide approximately 1.5 mls of lysed cell supernatant that can be used for subsequent analyses. Inoculate 2 ml of Luria Broth (plus antibiotic) with an appropriate E. coli strain (i.e. BL21 DE3) containing an expression plasmid encoding the protein of interest. Incubate the culture at 37°C with shaking overnight. Inoculate the 2ml overnight culture into 40 mls of LB (plus antibiotic) and incubate until mid-log phase of growth (A600 = 0.4 - 0. 6). This step normally takes less than 2 hours. Add IPTG to 0.5 mM and incubate the culture for an additional 4 hours or more. Harvest the cells by centrifugation and then resuspend the cell pellet with 1.8 mls of buffer (50 mM TrisHCl, pH 7.5 or any other suitable buffer). Transfer 0.6 mls of the cell suspension to a 2 ml microtube and add 0.2 gms of 0.1 mm disruptor beads. Using greater quantities of beads (up to 0.5 gms) did not increase the efficiency of cell lysis. Close the tube and shake vigorously for 2 minutes with the TurboMix attachment to the Vortex-Genie or Disruptor Genie. Pellet the cells by centrifugation at maximum speed for 5 minutes in a microfuge. Take an aliquot of the supernatant for SDS-PAGE analysis and decant the rest of the supernatant into a new tube.

Yeast/Fungi Disruption:
Disruptor Beads, 0.5 mm diameter, are recommended for disruption of yeast or fungi samples. A typical sample ratio would be 50% Disruptor Beads to 50% of yeast or fungus suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and yeast or fungus suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Yeast cells and fungi are generally more difficult to shear than bacterial cells, so increased disruption times may be necessary. Disruption in a cold room with chilled materials for 5 to 7 minutes at highest speed should be sufficient to disrupt the cell sample. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Soil Sample Disruption:
Either size of Disruptor Beads can be used for soil samples. A typical sample ratio would be 50% Disruptor Beads to 50% soil sample suspension by volume. Allow head space (~20%) within the microtube to facilitate disruption action. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Cell Disruption Using Glass Bead Media

Spherical lead free soda lime glass beads are commonly used for mechanical disruption of many yeast, bacterial and soil samples. Glass beads of a pre-determined size and volume are placed in a 1.5ml or 2.0ml microtube along with a pre-determined sample amount. The closed tube is then shaken vigorously at high speed, causing collisions between the glass beads and sample material. Scientific Industries’ Disruptor Genie® and TurboMix™ attachment for the Vortex-Genie 2 Family of Mixers are excellent choices for this process as they both simultaneously agitate and vortex at high speed, dramatically increasing cell or sample disruption. Each can hold up to twelve 1.5 ml or 2.0 ml microtubes at once. The disrupted cells may be removed after shaking for downstream processing.

Scientific Industries’ Disruptor Beads are packaged as 375g (8 fl. oz.) bottles in two sizes:

• 0.1 mm diameter beads (Catalog No. SI-BG01) — For use with Bacteria
• 0.5 mm diameter beads (Catalog No. SI-BG05) — For use with Yeast/Fungi

Care And Cleaning

Pre-preparation steps for Scientific Industries’ Disruptor Beads are generally unnecessary. If desired, they may be soaked in a 1:8 dilution of household bleach for 20 minutes, rinsed with copious amounts of distilled or RO water, and baked at 50 to 65°C for a minimum of 2 hours, or until completely dry. If the glass beads do not pour freely, repeat the cleaning and drying process. Disruptor Beads may also be autoclaved after proper disinfecting or cleaning.

The Disruptor Beads may be reused, if desired, after proper disinfecting or cleaning and autoclaving. Subsequent uses and excessive handling of the beads may result in the creation of fines, which could adversely affect cell disruption efficiency. As such, it is not recommended to frequently reuse Disruptor Beads.

Disruptor Beads may be stored at room temperature or frozen in an airtight container prior to use. In addition, the Disruptor Genie and TurboMix attachment for the Vortex-Genie 2 Family of Mixers may be used in cold rooms.

Sample Application Methods

NOTE: DETAILED DIRECTIONS FOR USE WILL DIFFER DEPENDING ON THE INDIVIDUAL PROTOCOL USED OR THE OUTCOME DESIRED. THE SAMPLE METHODS BELOW ARE EXAMPLES ONLY.

Bacteria Disruption:
Disruptor Beads, 0.1 mm diameter, are recommended for disruption of bacterial samples. A typical sample ratio would be 50% Disruptor Beads to 50% bacterial suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and bacterial suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Disruption at room temperature using chilled materials for 3 to 5 minutes at highest speed should be sufficient to recover 85% of the bacterial RNA. Disruption can be performed in a cold room as well. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Protocol for protein expression in Escherichia coli from a T7 expression system:
The following protocol is designed to provide approximately 1.5 mls of lysed cell supernatant that can be used for subsequent analyses. Inoculate 2 ml of Luria Broth (plus antibiotic) with an appropriate E. coli strain (i.e. BL21 DE3) containing an expression plasmid encoding the protein of interest. Incubate the culture at 37°C with shaking overnight. Inoculate the 2ml overnight culture into 40 mls of LB (plus antibiotic) and incubate until mid-log phase of growth (A600 = 0.4 - 0. 6). This step normally takes less than 2 hours. Add IPTG to 0.5 mM and incubate the culture for an additional 4 hours or more. Harvest the cells by centrifugation and then resuspend the cell pellet with 1.8 mls of buffer (50 mM TrisHCl, pH 7.5 or any other suitable buffer). Transfer 0.6 mls of the cell suspension to a 2 ml microtube and add 0.2 gms of 0.1 mm disruptor beads. Using greater quantities of beads (up to 0.5 gms) did not increase the efficiency of cell lysis. Close the tube and shake vigorously for 2 minutes with the TurboMix attachment to the Vortex-Genie or Disruptor Genie. Pellet the cells by centrifugation at maximum speed for 5 minutes in a microfuge. Take an aliquot of the supernatant for SDS-PAGE analysis and decant the rest of the supernatant into a new tube.

Yeast/Fungi Disruption:
Disruptor Beads, 0.5 mm diameter, are recommended for disruption of yeast or fungi samples. A typical sample ratio would be 50% Disruptor Beads to 50% of yeast or fungus suspension by volume. This ratio may be adjusted as necessary. Allow head space (~20%) within the microtube to facilitate disruption action. It is recommended that beads and yeast or fungus suspension be chilled prior to disrupting in order to offset any temperature rise within the microtube. Yeast cells and fungi are generally more difficult to shear than bacterial cells, so increased disruption times may be necessary. Disruption in a cold room with chilled materials for 5 to 7 minutes at highest speed should be sufficient to disrupt the cell sample. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.

Soil Sample Disruption:
Either size of Disruptor Beads can be used for soil samples. A typical sample ratio would be 50% Disruptor Beads to 50% soil sample suspension by volume. Allow head space (~20%) within the microtube to facilitate disruption action. Samples should not be run for longer than 10 minutes consecutively to avoid any temperature rise.