4110065Aqxp 9252007 239 PM Page 5 Brilliant Blue G250 dye to proteins Bradford1976 The dye exists in three forms cationicred neutral green and anionic blue Comptonand Jones 1985 ID: 936703
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Table of ContentsSection 1Introduction11.1Principle11.2Selecting a Protein Standard51.3Product Description9Section 2Instructions112.1Standard Assay Protocol112.2Microassay Protocol14Section 3Data Analysis18Section 4FAQs and Troubleshooting22Section 5O
rdering Information26Section 6References28Section 7Appendix30 4110065A.qxp 9/25/2007 2:39 PM Page 5 Brilliant Blue G-250 dye to proteins (Bradford1976). The dye exists in three forms: cationic(red), neutral (green), and anionic (blue) (Comptonand J
ones 1985).Under acidic conditions, thedye is predominantly in the doubly protonated red= 470 nm). However, when thedye binds to protein, it is converted to a stableunprotonated blue form (Aet al. 1975, Fazekes de St. Groth et al. 1963,Sedmack and Gro
ssberg 1977). It is this blue protein-dye form that is detected at 595 nm in theassay using a spectrophotometer or microplatereader. 470 nm (red)650 nm (green)595 nm (blue) 2 4110065A.qxp 9/25/2007 2:39 PM Page 8 protein buffers, stabilize the gree
n neutral dyespecies by direct binding or by shifting the pHNevertheless, many chemical reagents do notdirectly affect the development of dye color whenused in the standard protocol and the more common reagents are listed in Table 1. Themicroassay is
compatible with lower concentra-tions of reagents than listed in Table 1 due tothe larger sample volume-to-dye ratio. Since everyprotein-chemical reagent combination has noted reagents interfere in combination with certainproteins. However, with respe
ct to proteins such 4 4110065A.qxp 9/25/2007 2:39 PM Page 10 With the Quick Start Bradford protein assay, dyecolor development is significantly greater withBSA than with most other proteins, including -globulin. Therefore, the BSA standardwould be
an appropriate standard if the samplecontains primarily albumin, or if the protein beingassayed gives similar response to the dye. For acolor response that is typical of many proteins,globulin standard is appropriate. 6 4110065A.qxp 9/25/2007 2:39 P
M Page 12 Coomassie is a trademark of Imperial Chemical Industries. Triton is a trademarkof Union Carbide Corp. Tween is a trademark of ICI Americas, Inc. *The concentration limits for compatibility with the microassay are values in Table 1. TBP, 5 m
MTBS (25 mM Tris, 0.15 M NaCl,TCEP, 20 mM Thio-urea, 1 MTricine, pH 8, 50 mMTriethanolamine, pH 7.8, 50 mMTris, 1 MTris-glycine (25 mM Tris, 192 mMTris-glycine-SDS, (25 mM Tris,Triton X-100, 0.05% Tween 20, 0.01%Urea, 4 MPMSF, 2 mMSodium carbonate, 0.
1 MSodium chloride, 2.5 MSodium hydroxide, 0.1 MSucrose, 10% 4110065A.qxp 9/25/2007 2:39 PM Page 14 Bovine Serum Albumin Standard Set:0.5, 0.25, 0.125 mg/ml) in 2 ml tubes.Bovine Gamma-Globulin Standard Set:Set of 7 concentrations of gamma-globuli
n (2,1.5, 1, 0.75, 0.5, 0.25, 0.125 mg/ml) in 2 ml 4110065A.qxp 9/25/2007 2:39 PM Page 16 appendix as a guide for diluting the proteinstandard. (The dilutions in the tables areenough for performing triplicate measure-ments of the standards.) For th
e diluent, usethe same buffer as in the samples (refer toTroubleshooting section for more informa-tion). Protein solutions are normally assayedin duplicate or triplicate. For convenience,the BSA or gamma-globulin standard setsreagent. 4.Pipet each st
andard and unknown samplemicroplate wells (the 1 ml assay may be 12 4110065A.qxp 9/25/2007 2:39 PM Page 18 6.Set the spectrophotometer to 595 nm. Zerorequired for microplate readers). Measure theabsorbance of the standards and unknownsamples. Refe
r to Section 3 for data analysis. If the spectrophotometer has a reference and sample holder, the instrumentcan be zeroed with two blank samples. If the effect of buffer on absorbance is required,zero the instrument with a cuvette filled withwater an
d dye reagent in the reference holder. 2.2 Microassay Protocol1.The microassay protocol can be performed intwo different formats, a 2 ml cuvette assay 14 4110065A.qxp 9/25/2007 2:39 PM Page 20 4.Pipet each standard and unknown sampledisposable cuv
ettes, or microplate wells. Add1x dye reagent to each tube or cuvette andvortex: for microplates, mix the samplesusing a microplate mixer. Alternatively, use areagent. Depress and release the plungerrepeatedly to mix the sample and reagent inreagent t
o the next set of wells.AssayVolume ofVolume ofStandard and Sample1x Dye Reagent2 ml1 ml1 mlMicroplate150 µl150 µl 16 4110065A.qxp 9/25/2007 2:39 PM Page 22 1.If the spectrophotometer or microplate readerwas not zeroed with the blank, then averageb
lank value from the standard and unknown2.Create a standard curve by plotting the sample concentration using the standardcurve. If the samples were diluted, adjust theby multiplying by the dilution factor used. 18 4110065A.qxp 9/25/2007 2:39 PM P
age 24 20 Fig 1. Typical standard curves using the standard 5 mlprocedure with BSA and gamma-globulin standards. 4110065A.qxp 9/25/2007 2:39 PM Page 26 21 Fig 2. Typical standard curves using the microassayprocedure with BSA and gamma-globulin s
tandards. 4110065A.qxp 9/25/2007 2:39 PM Page 27 22 FAQs and Troubleshooting1 The buffer that I normally use isnot in the list of compatiblereagents. How will I know if itinterferes with the Quick StartBradford assay?It is best to run two standard
curves,one with protein in the same buffer asyour sample and one with protein inwater, and then plot a graph of protein concentration versusabsorbance. If the buffer does notinterfere, the two standard curves willhave identical slopes. Adding thebuff
er or interfering component to the standards used to construct the standard curve for the actual proteininterference. 4110065A.qxp 9/25/2007 2:39 PM Page 28 24 4 Does the binding of the bluedye to cuvettes skew results?Bio-Rad's disposable polysty
renecuvettes (catalog #223-9950) arerecommended for the protein assay.amount of dye that binds to them isnegligible (Bradford 1976). Therefore,removal of the residual blue colorbetween each sample reading isunnecessary. However, since theprocedures, t
here are several recom-mended treatments for dye removal: Rinse the cuvette with methanol, Rinse the cuvette with glasswaredetergent, followed by ddi water, Soak the cuvette in 0.1 N HCl forwater. 4110065A.qxp 9/25/2007 2:39 PM Page 30 26 Orderi
ng InformationCatalog #Description500-0201Quick Start Bradford Protein Assay Kit 1, includes 1 L 1x dye reagent and 5 x 2 ml bovine serum albumin standard at 2 mg/ml500-0202Quick Start Bradford Protein Assay Kit 2, includes 1x dye reagent (1 L), bovin
e serum albumin standardset (2 sets of 7 standards, 0.1252.0 mg/ml, 2 ml)500-0203Quick Start Bradford Protein Assay Kit 3, includes 1x dye reagent (1 L), bovine -globulin standard (5 x 2 mg/ml)500-0204Quick Start Bradford Protein Assay Kit 4, include
s 1 L 1x dye reagent and bovine gamma globulinstandard set with 2 x 2 ml each concentration500-0205Quick StartBradford 1 x Dye Reagent, 1L500-0206 Quick StartBovine Serum Albumin Standard, 4110065A.qxp 9/25/2007 2:39 PM Page 32 28 ReferencesBradfo
rd MM, A rapid and sensitive method for the quantitationof microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem, 72, 248254 (1976)Compton SJ and Jones CG, Mechanism of dye response andinterference in the Bradfo
rd protein assay, Anal Biochem 151,Fanger B, Adaptation of the Bradford protein assay to membrane-bound proteins by solubilizing in glucopyranosideFazekas de St. Groth S et al., Two new staining procedures forquantitative estimation of proteins on ele
ctrophoretic strips,perchloric acid solution for staining in electrophoresis and isoelectric 4110065A.qxp 9/25/2007 2:39 PM Page 34 5 ml Standard AssayStandardSource ofDiluentFinalTube #Volume (µl)StandardVolume (µl)[Protein] (µg/ml)1300 2 mg/ml
stock0 2,000 2375 2 mg/ml stock125 1,500 3325 2 mg/ml stock325 1,000 4175 Tube 2175 750 5325 Tube 3325 500 6325 Tube 5325 250 7325 Tube 6325 125 8 (blank)325 0 1 ml Standard AssayStandardSource ofDiluentFinalTube #Volume (µl)StandardV
olume (µl)[Protein] (µg/ml)170 2 mg/ml stock0 2,000275 2 mg/ml stock25 1,500370 2 mg/ml stock70 1,000435 Tube 235 750570 Tube 370 500670 Tube 570 250770 Tube 670 125 8 (blank)70 0 4110065A.qxp 9/25/2007 2:39 PM Page 36 2 ml Microas
say Cuvette Dilutions for BSA orGamma-Gobulin Standard SetsStandardSource ofDiluentFinalTube #Volume (µl)StandardVolume (µl)[Protein] (µg/ml)140 2 mg/ml3,160 25 235 2 mg/ml3,465 20335 1.5 mg/ml3,465 15 435 1 mg/ml3,465 10535 0.5 mg/ml3,465 5 6
35 0.25 mg/ml3,465 2.5 735 0.125 mg/ml3,465 1.25 8 (blank)3,200 0 Microplate Microassay Dilutions for StandardSource ofDiluentFinalTube #Volume (µl)StandardVolume (µl)[Protein] (µg/ml)110 2 mg/ml stock790 25 210 2 mg/ml stock990 20 36 2 mg/
ml stock794 15 4500 Tube 2500 10 5500 Tube 4500 5 6500 Tube 5500 2.5 7500 Tube 6500 1.25 8 (blank)500 0 4110065A.qxp 9/25/2007 2:39 PM Page 38 BradfordProtein AssayFor technical service call your local Bio-Rad office, or in the US, 41100