To alleviate the frustration of discovering these incompatibilities by trial and error, our technical support scientists have characterized the effects of commonly ...
TECH TIP #68
Protein assay compatibility table TR0068.5
Introduction No single protein assay method is compatible with all sample components that might be encountered in typical laboratory research. To alleviate the frustration of discovering these incompatibilities by trial and error, our technical support scientists have characterized the effects of commonly used buffers and sample components on the behavior of seven different Thermo Scientific Pierce* Protein Assays. Results of these compatibility experiments are summarized in tabular form on the following two pages. The table serves as a guide for assessing which protein assay(s) might be most effective with a given sample type. Use this information as a guide only. It is the researcher’s responsibility to validate the compatibility of specific sample buffers. Even minor changes in buffers and components affect each assay method to some extent. Researchers must decide if these effects are acceptable for their specific purposes.
About the Compatibility Table The table indicates the maximum compatible concentrations for substances tested with the Test Tube procedure on the midpoint concentration of BSA standard (i.e., 1000µg/mL for most assays; 10µg/mL for the Micro BCA Assay). Substances were considered compatible at the indicated concentration if the error in protein concentration estimation caused by the presence of the substance in the sample was less than or equal to 10%.
Tested Thermo Scientific Pierce Protein Assays.
Notes and Symbols: • • • • • • • • •
Part No.
Name
22662
660 nm
23225
BCA
23235
Micro BCA
23250
BCA – Reducing Agent Compatible (BCA-RAC)
23252
Microplate BCA-RAC
23240
Modified Lowry
23236
Coomassie Plus (Bradford)
23200
Coomassie (Bradford)
Compounds are listed alphabetically using common names or abbreviations, except sodium compounds which are alphabetized under “Na”. Dilutions are expressed as “neet” (= undiluted) or in the form of a ratio, where “1:2” means 2-fold dilution. n/a Denotes that the compound was not tested in this assay. Ø Denotes compounds that were not compatible at the lowest concentration tested. * Thermo Scientific product trademark; “PER Reagents” are Thermo Scientific Pierce Cell Lysis Reagents. ** Trademarks that are the property of other companies; see end of document. † Value when the 660nm Assay is run using the ionic detergent compatibility reagent (IDCR, Part No. 22663). †† Selected values for the regular BCA-RAC Kit are given in parentheses in the column for the Microplate BCA-RAC. ‡ Compound (buffer) whose formulation is described more fully in the following table: Part No. 28390 28374 28382 28388 28386 28372 89900 28379 28380 28378 28398
Buffer
Formulation
2-D sample buffer Laemmli SDS sample buffer MES-buffered saline pH 4.7 Modified Dulbecco’s PBS Na carb-bicarb pH 9.4 Na citrate-carbonate pH 9 Na citrate-MOPS pH 7.5 Phosphate-buffered saline (PBS) RIPA Buffer Tris-buffered saline (TBS) Tris-glycine pH 8.0 Tris-glycine-SDS pH 8.3 Tris-HEPES-SDS
(8M urea, 4% CHAPS) or (7M urea, 2M thiourea, 4% CHAPS) 65mM Tris-HCl, 10% glycerol, 2% SDS, 0.025% bromophenol blue 0.1M MES, 150mM NaCl pH 4.7 8mM sodium phosphate, 2mM potassium phosphate, 0.14M NaCl, 10mM KCl, pH 7.4 0.2M sodium carbonate-bicarbonate pH 9.4 0.6M sodium citrate, 0.1M sodium-carbonate pH 9 0.6M sodium citrate 0.1M MOPS pH 7.5 100mM sodium phosphate, 150mM NaCl pH 7.2 50mM Tris, 150mM NaCl, 0.5% DOC, 1% NP40, 0.1% SDS pH 8.0 25mM Tris, 150mM NaCl pH 7.6 25mM Tris, 192mM glycine pH 8.0 25mM Tris, 192mM glycine, 0.1% SDS pH 8.3 100mM Tris, 100mM HEPES, 3mM SDS
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Thermo Scientific Pierce Protein Assay Test Compound
660 nm
BCA
Micro BCA
††Microplate BCA-RAC
Coomassie Plus
Coomassie
Modified Lowry
2-D sample buffer ‡ 2-Mercaptoethanol ACES, pH 7.8 Acetone Acetonitrile Ammonium sulfate Aprotinin Ascorbic acid Asparagine Bicine Bis-Tris pH 6.5 Borate (50mM) pH 8.5 B-PER* Reagent B-PER Reagent II B-PER Reagent PBS Brij**-35 Brij-56 Brij-58 Bromophenol blue (in 50mM NaOH) Calcium chloride (in TBS pH 7.2) Cesium bicarbonate Cetylpyridinium chloride CHAPS CHAPSO CHES Cobalt chloride (in TBS pH 7.2) CTAB Cysteine Dithioerythritol (DTE) Dithiothreitol (DTT) DMF DMSO DTAB EDTA EGTA EPPS pH 8.0 Ethanol Ferric chloride (in TBS pH 7.2) Glucose Glutathione (reduced) Glycerol (fresh) Glycine-HCl pH 2.8 Guanidine-HCl HEPES pH 7.5 Hydrides (Na2BH4 or NaCNBH3) Hydrochloric acid (HCl) Imidazole pH 7.0 I-PER* Reagent Laemmli SDS sample buffer ‡ Leupeptin Mannitol Melibiose Mem-PER* Reagent MES-buffered saline pH 4.7 ‡ MES pH 6.1 Methanol Magnesium chloride Modified Dulbecco's PBS ‡ MOPS pH 7.2 M-PER* Reagent
neet 1M 50mM 50% 50% 125mM 2mM 500mM 40mM >1M 50mM neet 1:2 1:2 1:2 5% n/a 5% 0.031% 40mM 100mM 2.5% † 5% 4% >500mM 20mM 2.5% † 350mM 25mM 500mM 50% 50% 2% † 20mM 20mM 200mM 50% 5mM 500mM 100mM 50% 100mM 2.5M 100mM Ø 125mM 200mM 1:4 neet † 80µM 100mM 500mM neet neet 125mM 50% >1M neet 125mM 1:2
n/a 0.01% 25mM 10% 10% 1.5M 10mg/L Ø 1mM 20mM 33mM neet neet n/a n/a 5% 1% 1% Ø 10mM 100mM n/a 5% 5% 100mM 0.8mM n/a Ø 1mM 1mM 10% 10% n/a 10mM Ø 100mM 10% 10mM 10mM n/a 10% 100mM 4M 100mM Ø 100mM 50mM neet Ø 10mg/L n/a Ø neet neet 100mM 10% n/a neet 100mM neet
n/a 1mM 10mM 1% 1% Ø 1mg/L Ø n/a 2mM 0.2mM 1:4 n/a n/a n/a 5% 1% 1% Ø 10mM 100mM n/a 1% 5% 100mM Ø n/a Ø Ø Ø 1% 1% n/a 0.5mM Ø 100mM 1% 0.5mM 1mM n/a 1% n/a 4M 100mM Ø 10mM 12.5mM n/a Ø 10mg/L n/a n/a neet 1:4 100mM 1% n/a neet 100mM n/a
n/a 25mM (35) Ø Ø 30% Ø Ø n/a Ø 1mM 16.5mM Ø 1:3 1:4 1:4 0.63% n/a 0.50% Ø 1mM Ø n/a 10% (10) Ø 50mM 0.4mM n/a 2.5mM 2.5mM 5mM (5) 5% 0.25% n/a 5mM (20) 5mM (10) Ø Ø 5mM Ø 10mM 5% 50mM 1.5M (2) 200mM (200) n/a Ø 30mM (50) n/a Ø Ø n/a n/a 1:2 Ø 100mM (100) 0.5% 100mM neet 200mM 1:2
n/a 1M 100mM 10% 10% 1M 10mg/L 50mM 10mM 100mM 100mM neet 1:2 1:4 n/a 0.062% 0.031% 0.016% Ø 10mM 100mM n/a 5% 5% 100mM 10mM n/a 10mM 1mM 5mM 10% 10% n/a 100mM 2mM 100mM 10% 10mM 1M n/a 10% 100mM 3.5M 100mM n/a 100mM 200mM n/a Ø 10mg/L n/a 100mM neet neet 100mM 10% n/a neet 100mM neet
n/a 1M 100mM 10% 10% 1M 10mg/L 50mM 10mM 100mM 100mM neet 1:2 n/a n/a 0.125% 0.031% 0.031% Ø 10mM 100mM n/a 5% 5% 100mM 10mM n/a 10mM 1mM 5mM 10% 10% n/a 100mM 2mM 100mM 10% 10mM 1M n/a 10% 100mM 3.5M 100mM n/a 100mM 200mM n/a Ø 10mg/L n/a 100mM n/a neet 100mM 10% n/a neet 100mM n/a
n/a 1mM n/a 10% 10% Ø 10mg/L 1mM 5mM n/a n/a n/a n/a n/a n/a 0.031% 0.062% 0.062% Ø n/a 50mM n/a 0.062% 0.031% n/a n/a n/a 1mM Ø Ø 10% 10% n/a 1mM 1mM n/a 10% n/a 100mM n/a 10% 100mM n/a 1mM n/a 100mM 25mM n/a Ø 10mg/L n/a 25mM n/a n/a 125mM 10 % n/a n/a n/a n/a
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Thermo Scientific Pierce Protein Assay
Continued… Test Compound n-Acetylglucosamine Na acetate pH 4.8 Na azide Na bicarbonate Na carb-bicarbonate pH 9.4 ‡ Na chloride Na citrate pH 4.8 Na citrate-carbonate pH 9 ‡ Na citrate-MOPS pH 7.5 ‡ Na deoxycholate (DOC) Na hydroxide (NaOH) Na phosphate NE-PER* Reagent (CER) NE-PER Reagent (NER) Nickel chloride (in TBS pH 7.2) NP-40 Octyl beta-glucoside Octylthioglucoside Na-orthovanadate (in PBS pH 7.2) Phenol Red Phosphate-buffered saline (PBS) ‡ PIPES pH 6.8 PMSF in isopropanol Potassium thiocyanate P-PER Reagent RIPA buffer ‡ SDS Sodium compounds Span** 20 Sucrose TCEP Thimerosal Thiourea TLCK TPCK T-PER* Reagent Tricine pH 8.0 Triethanolamine pH 7.8 Tris-buffered saline (TBS) ‡ Tris-glycine pH 8.0 ‡ Tris-glycine-SDS pH 8.3 ‡ Tris-HCl pH 8.0 Tris-HEPES-SDS ‡ Triton** X-100 Triton X-114 Triton X-305 Triton X-405 Tween** 20 Tween 60 Tween 80 Urea Y-PER* Reagent Y-PER Plus Reagent Zinc chloride (in TBS pH 7.2) Zwittergent** 3-14
660 nm 100mM 100mM 0.125% 100mM 1:3 1.25M 12.5mM Ø 1:16 0.25% 125mM 500mM neet neet 10mM 5% 5% 10% 50mM 0.5mg/mL neet 100mM 1mM 250mM 1:2 neet 0.01%, 5% † (see Na) n/a 50% 40mM 0.25% 2M 5mg/mL 4mg/mL 1:2 500mM 100mM neet neet neet † 250mM neet † 1% 0.50% 9% 5% 10% 5% 5% 8M Ø 1:2 10mM 0.05%
BCA 10mM 200mM 0.2% 100mM neet 1M 200mM 1:8 1:8 5% 100mM 100mM neet neet 10mM 5% 5% 5% 1mM Ø neet 100mM 1mM 3M Ø neet 5% (see Na) 1% 40% Ø 0.01% n/a 0.1mg/L 0.1mg/L 1:2 25mM 25mM neet 1:3 neet 250mM n/a 5% 1% 1% 1% 5% 5% 5% 3M neet neet 10mM 1%
Micro BCA Ø 200mM 0.20% 100mM neet 1M 5mM 1:600 1:600 5% 50mM 100mM n/a n/a 0.2mM 5% 0.1% 5% 1mM Ø neet 100mM 1mM n/a n/a 1:10 5% (see Na) 1% 4% Ø Ø n/a 0.1mg/L 0.1mg/L n/a 2.5mM 0.5mM 1:10 1:10 neet 50mM n/a 5% 0.05% 1% 1% 5% 0.5% 5% 3M n/a n/a 0.5mM Ø
††Microplate BCA-RAC Ø Ø 0.01% Ø neet 150mM 50mM Ø neet n/a Ø 100mM 1:2 1:4 Ø Ø 2.5% (10) 7% 0.5mM 3.125µg/mL neet 25mM 0.125mM Ø 1:2 1:2 5% (10) (see Na) n/a 40% (40) 10mM (10) 0.03% n/a Ø Ø n/a 0.5mM 25mM neet Ø Ø 35mM (50) n/a 7% (10) 2% (2) 1% Ø 10% (10) 5% 2.5% 3M (4) n/a n/a Ø 2% (2)
Coomassie Plus 100mM 180mM 0.5% 100mM neet 5M 200mM neet n/a 0.4% 100mM 100mM 1:4 neet 10mM 0.5% 0.5% 3% 1mM 0.5mg/mL neet 100mM 1mM 3M Ø 1:40 0.016% (see Na) 0.5% 10% 125mM 0.01% n/a 0.1mg/mL 0.1mg/mL neet 100mM 100mM neet neet 1:4 2M n/a 0.062% 0.062% 0.125% 0.025% 0.031% 0.025% 0.016% 3M n/a neet 10mM 0.025%
Coomassie 100mM 180mM 0.5% 100mM neet 5M 200mM neet neet 0.05% 100mM 100mM n/a n/a 10mM 0.5% 0.5% 3% 1mM 0.5mg/mL neet 100mM 1mM 3M Ø 1:10 0.125% (see Na) 0.5% 10% 125mM 0.01% n/a 0.1mg/L 0.1mg/L n/a 100mM 100mM neet neet 1:2 2M n/a 0.125% 0.125% 0.5% 0.5% 0.062% 0.1% 0.062% 3M n/a n/a 10mM 0.025%
Modified Lowry n/a 200mM 0.2% 100mM n/a 1M n/a n/a n/a n/a 100mM 100mM n/a n/a n/a 0.016% 0.031% n/a n/a n/a n/a n/a 1mM 100mM Ø n/a 1% (see Na) 0.25% 7.5% n/a 0.01% n/a 0.01mg/L 0.1mg/L n/a n/a n/a n/a n/a n/a 10mM n/a 0.031% 0.031% 0.031% 0.031% 0.062% n/a 0.031% 3M n/a n/a n/a n/a
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