2012_10_22 Protein Solution Recipe Comparisons
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Tris pH 8.8 (150 mL)gMWmolesM2012_12_20: make 100 mL of 4x protein buffer using Amanda's RecipeCompare to BioRad Cat #161-073725050
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Tris27.23121.140.22478124481.498541632ItemAmtAmt to USE 12/21/2012
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glycerol (mL)50<-- make it 10X glycerol when 1x to match BioRad -->40
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SDS (g)10commercial 4X has 1% LDS(?), so stick with Amanda's10
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1M Tris-HCl pH 6.831.25<-- commercial 4X buffer is 250mM so use 50 mL of 0.5M -->50
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bromophenol blue slurry (5mg/mL in water; vortex before using) uL1000<-- use 5 mg instead of making a solution first. Amanda's was dark enough. The commercial recipe lists 0.005% Bromophenol Blue5mg
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0.125H2ONOTE: I added more bromphenol blue. About double would have been ideal.
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total volume (mL)92.25
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Fill to 100 mL
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Can we do BCA?1x form:
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% difference:
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LOADING BUFFERAmanda (4x)Amanda: scaled to 9.5 mLBioRad commercial buffer (2x):BioRad 4X Laemmli Sample Buffer (Cat # 161-0747)official BioRad (~ 3x)Biorad: conc scaled to 4x:100*(Amanda-BioRad)/BioRadLOADING BUFFERofficial BioRad (~ 3x)BCA assa limits: http://www.piercenet.com/instructions/2161296.pdf
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glycerol (mL)44.75200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% Coomassie12.53.3343glycerolglycerol (mL)2.50.838.77% glycerol10%
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SDS (g)0.80.95= 8 mL of 10% SDS solution(catalog #161-0739) Brilliant Blue G-250, 2% ß-mercaptoethanol or 350 mM DTT (added fresh)0.110% SDS solution:22.670.27g SDS256SDSSDS (g)20.677.02% SDS. Note: this is ABOVE THE 5% LIMIT for BCA: http://www.piercenet.com/instructions/2161296.pdf5%
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1M Tris-HCl pH 6.82.52.968750.5 M Tris-HCl, pH 6.8 3.75 ml 1.251.6778Tris-HCl pH 6.81M Tris-HCl pH 6.81.250.420.42M Tris-HCl, pH 6.8
250 mM. Note: they don't say pH adjusted but this is a reasonable pH.
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bromophenol blue slurry (5mg/mL in water; vortex before using) uL809550% Glycerol 15.0 ml 200266.67-645 mg/mL dye solutionbromophenol blue slurry (5mg/mL in water; vortex before using) uL20066.67
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H2O1.0% Bromophenol blue 0.3 ml 3.551.57H2O3.551.18
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10% SDS 6.0 ml
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total volume (mL)89.5diH2O to 30 ml 109.59.59.5
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Add b-mercap
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STAINING
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AmandaAmandaOnline Recipe 1:Online 2Janet's prep 10/22/2012
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MeOH80200400450300
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water280700500450600
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acetic acid40100100100100100% weight/volume1g/mL1000mg/mL
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dye (g)0.4110.5<-- 0.05% w/(total volume)1used R form of dye from Sigma1% weight/volume0.01g/mL10mg/mL
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TOTAL4001,0001,0001,0001,0000.5% weight/volume0.005g/mL5mg/mL
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0.05% weight/volume0.0005g/mL0.5mg/mLOR0.5g/L
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http://www.cytographica.com/lab/solutions/Coomassie.htmhttp://www.case.edu/med/coller/Coller%20Protocol%20Book.pdf
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AMANDA
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DESTAINING:
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acetic acid10050
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methanol2000
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ethanol050
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water70090010% acetic acid, and destained with a 5% ethanol/5% acetic acid solution.
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TOTAL1000
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For 10 L
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Electrode BuffergMWmolesM
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Tris30121.140.24764735020.02476473502
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Glycine14475.06661.9182965530.1918296553
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SDS (optional: I didn't add 10/2012)10
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