Search
Search
View additional product information for Pierce™ Bradford Protein Assay Kit - FAQs (23200)
40 product FAQs found
The Pierce Bradford Protein Assay Kit is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended. Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Sorry, the Pierce Bradford Protein Assay Reagent is only available as a component of the Pierce Bradford Protein Assay Kit.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
Each of the commonly used total protein assay methods exhibits some degree of varying response toward different proteins. These differences relate to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein’s color response. Most protein assay methods use BSA or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined. However, if great accuracy is required, prepare the standard curve from a pure sample of the target protein.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
You can test the tolerance of the assay for your specific buffer formulation. For in-house generated compatibility information, substances were considered compatible at the indicated concentration in the Standard Test Tube Protocol (found in the manual for each protein assay) if the error in protein concentration estimation caused by the presence of the substance was less than or equal to 10%. The substances were tested using WR prepared immediately before each experiment. Blank-corrected 562nm absorbance measurements (for a 1000µg/mL BSA standard + substance) were compared to the net 562nm measurements of the same standard prepared in 0.9% saline.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
It is possible to have a substance additive affect such that even though a single component is present at a concentration below its listed compatibility, a sample buffer containing a combination of substances could interfere with the assay. You should take steps to eliminate or minimize the effects of the interfering substance(s) by diluting or removing the substance.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Each protein assay method has its particular sensitivities. Generally, the following substances should be avoided for the given protein assay method:
Several strategies exist for overcoming or eliminating sample incompatibility with protein assays. The simplest method is to assay the sample after diluting it several-fold in a compatible buffer. If the starting concentration of protein is sufficient to remain within the protein assay working range upon its dilution, then this method will often reduce the amount of interfering substance in the sample to the point where it no longer interferes. Another method is to dialyze or desalt samples into a buffer that is compatible with the assay.
Precipitation can be used to eliminate interfering substances. After causing the protein to precipitate with acetone or trichloroacetic acid (TCA), the supernatant containing the interfering substance can be removed. Then the protein pellet is dissolved in the assay working reagent, and the protein assayed performed as usual. A general protocol for protein precipitation is provided in our Tech Tip.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Refer to the information in the product-specific instruction booklet or our Tech Tip: Protein Quantitation Assay Compatibility Table (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Often, an alternative wavelength can be used, although the slope of the standard curve and the overall assay sensitivity will most likely be reduced. Our Tech Tip (https://tools.thermofisher.com/content/sfs/brochures/TR0025-Protein-assay-spectra.pdf) offers additional information on determining acceptable wavelengths for measuring protein assays.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Each method has its particular sensitivities. Apart from the information presented in the product-specific instruction booklet, we offer an expanded protein assay compatibility table in this Tech Tip.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Several factors affect protein assay accuracy and precision:
Replicates: The only way to evaluate the extent of random error is to include replicates of each standard and test sample. Because all test samples are evaluated by comparison to the standard curve, it is especially important to run the standards in at least triplicate. The standard deviation (SD) and coefficient of variation (CV) can then be calculated, providing a degree of confidence in your pipetting precision. If replicates are used, curve-fitting is done with the average values (minus obvious outliers).
Blank correction: It is common practice to subtract the absorbance of the zero assay standard(s) from the all other sample absorbance values. However, if replicate zero-assay standards will be used to calculate error statistics, then another independent value may be required for blank-correction. If the standards were prepared in a buffer to match that of the test samples, and this buffer contains components that may interfere with the assay chemistry, it is informative to blank the absorbances with a "water reference" (i.e., a zero-protein, water sample). Differences between the water reference and zero standard sample are then indicative of buffer effects.
Standard curve slope: The standard curve slope is directly related to assay accuracy and sensitivity. All else being equal, the steepest part of the curve is the most reliable. For most protein assays, the standard curve is steepest (i.e., has the greatest positive slope) in the bottom half of the assay range. In fact, the upper limit of an assay range is determined by the point at which the slope approaches zero; the line there is so flat that even a tiny difference in measured absorbance translates to a large difference in calculated concentration.
Measurement wavelength: The measurement wavelengths that are recommended for each protein assay method are optimal because they yield standard curves with maximal slope. This usually, but not always, corresponds to the absorbance maximum. (In certain circumstances, other considerations are also important in choosing the best possible measurement wavelength, such as avoiding interference from sample components that absorb at similar wavelengths). In fact, for most protein assays, depending on the precision required, acceptable results can be obtained using any measurement wavelengths within a certain range.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
One situation in which the dilution factor is important to consider is when the original sample has been pre-diluted relative to the standard sample. Suppose the original protein sample is actually known to be approximately 5 mg/mL. This is too concentrated to be assayed by the Pierce Bradford Plus Protein Assay Kit, for example, whose assay range in the standard microplate protocol is 100-1500 µg/mL. However, you could dilute it 5-fold in buffer (i.e., 1 part sample plus 4 parts buffer) and then use that diluted sample as the test sample in the protein assay. If the test sample produces the same absorbance as the 1000 µg/mL standard sample, then you can conclude that the test (5-fold diluted) sample is 1000 µg/mL, and therefore the original (undiluted) sample is 5 x 1000 µg/mL = 5000 µg/mL = 5 mg/mL.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No. It is neither necessary nor helpful to know the protein concentration as it exists when the samples are diluted in assay reagent. The protein concentration when diluted by assay reagent is almost certainly not the value of interest; instead, one wants to know the protein concentration of the original test sample.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
No. Contrary to what many people assume, it is neither necessary nor even helpful to know the actual amount (e.g., micrograms) of protein applied to each well or cuvette of the assay. The amount of protein per well is almost certainly not the value of interest; instead, one usually wants to know the protein concentration of the original test sample.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Enter the concentration values for the standards in Column A and their corresponding absorbance data in Column B. Highlight both columns and from the Insert menu select Chart and XY (Scatter). Click on the resulting graph and select Add Trendline from the Chart menu. While viewing the graph next to the open Format Trendline window, choose Polynomial and set the Order to 2, 3 or 4 until the best-fit appears. Check the box near the bottom called Display Equation on Chart; then close the Format Trendline window. Use the resulting equation to determine protein concentration (y) of an unknown sample by inserting the sample’s absorbance value (x).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Most modern plate readers and spectrophotometers have associated software that automatically plots a linear or curvilinear regression line through the standard points, interpolates the test samples on that regression line, and reports the calculated value. However, there are different methods for making the calculations “by hand”. You can find a detailed explanation and example in our Tech Tip.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
With most protein assays, sample protein concentrations are determined by comparing their assay responses to that of a dilution-series of standards whose concentrations are known. The responses of the standards are used to plot or calculate a standard curve. Absorbance values of unknown samples are then interpolated onto the plot or formula for the standard curve to determine their concentrations. The most accurate results are possible only when unknown and standard samples are treated identically. This includes assaying them at the same time and in the same buffer conditions, if possible. Because different pipetting steps are involved, replicates are necessary if you wish to calculate statistics (e.g., standard deviation, coefficient of variation) to account for random error. It is imperative to run a new standard curve for each set of samples to be tested
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Simply multiply the calculated concentration of the diluted sample by the dilution factor. For example: A protein sample is known to be approximately 5 mg/mL. This is too concentrated to be assayed by the Pierce Bradford Plus Protein Assay Kit, whose assay range in the standard microplate protocol is 100-1500 µg/mL. However, you could dilute it 5-fold in buffer (i.e., 1 part sample plus 4 parts buffer) and then use that diluted sample as the test sample in the protein assay. If the test sample produces the same absorbance as the 1000 µg/mL standard sample, then you can conclude that the test (5-fold diluted) sample is 1000 µg/mL, and therefore the original (undiluted) sample is 5 × 1000 µg/mL = 5000 µg/mL = 5 mg/mL.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The unit of measure used to express the standards is by definition the same unit of measure associated with the calculated value for the unknown sample (i.e., final results for unknown samples will be expressed in the same unit of measure as was used for the standards). For example, if the standard concentrations are expressed as micrograms per milliliter, then the concentrations for the unknown samples, which are determined by comparison to the standard curve, are also expressed as micrograms per milliliter.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, we recommend storing at -20 degrees C and they will likely be good for 2-3 months, or about 2 freeze/thaws. Samples subjected to more than ˜2 freeze/thaw cycles may give variable results in most assays
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Protein standards should preferably be diluted using the same diluent as the sample(s). Sample assay responses are directly comparable to each other if they are processed in exactly the same manner. Variance in protein quantity is the only possible cause for differences in final absorbance (color intensity) if samples are dissolved in the same buffer and the same stock solution of assay reagent is used for all samples.
However, if only a “rough” estimate of protein concentration is needed, a blank-only correction can be used. In this case, a blank is prepared in the diluent of the sample to correct for its raw absorbance. The concentration of the sample is then determined from a standard curve obtained from a series of dilutions of the protein of known concentration prepared in water or saline solution.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We offer BSA and BGG as protein standards for protein assays.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Protein concentrations are generally determined and reported with reference to standards of a common protein, such as bovine serum albumin (BSA). If precise quantitation of an unknown protein is required, it is advisable to select a protein standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard may be used when assaying immunoglobulin samples.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Because proteins differ in their amino acid compositions, each one responds somewhat differently in each type of protein assay. Therefore, the best choice for a reference standard is a purified, known concentration of the most abundant protein in the samples. This is usually not possible to achieve, and it is seldom convenient or necessary. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve in the selected protein assay method and is readily available to any laboratory at any time. Generally, bovine serum albumin (BSA) works well as a protein standard because it is widely available in high purity and relatively inexpensive. Alternatively, bovine gamma globulin (BGG) is a good standard when determining the concentration of antibodies because BGG produces a color response curve that is very similar to that of immunoglobulin G (IgG).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Pierce Bradford Protein Assay Kit and Pierce Bradford Plus Protein Assay Kit are variations on the use of Coomassie G-250 dye as a colorimetric reagent for the detection and quantitation of total protein first reported by Bradford in 1976. The Thermo Scientific 660 nm Protein Assay is a dye-based reagent that offers the same convenience as Coomassie-based assays while overcoming several of their disadvantages. In particular, the 660 nm Assay is compatible with most detergents and produces a more linear response curve (the detailed assay chemistry is proprietary). Our fluorometric protein assays are also based on dye binding chemistries.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Most colorimetric protein assay methods can be divided into two groups based on the type of chemistry involved: those involving protein-copper chelation with secondary detection of the reduced copper and those based on protein-dye binding with direct detection of the color change associated with the bound dye.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Each protein in a sample responds uniquely in a given protein assay, and this protein-to-protein variation is observed as differences in the amount of color (absorbance) obtained when the same mass of various proteins is assayed concurrently by the same method. These differences in color response relate to differences in amino acid sequence, isoelectric point (pI), secondary structure, and the presence of certain side chains or prosthetic groups.
Depending on the sample type and purpose for performing an assay, protein-to-protein variation is an important consideration in selecting a protein assay method and in selecting an appropriate assay standard (e.g., BSA vs. BGG). Protein assay methods based on similar chemistry have similar protein-to-protein variation.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Before the sample is analyzed, it must be solubilized in a buffered aqueous solution. Depending on the source material and the procedures involved before performing the protein assay, the sample will likely contain a variety of non-protein components. Awareness of these components is critical for choosing an appropriate assay method and evaluating the cause of anomalous results. Every type of protein assay is adversely affected by substances of one sort or another. Components of a protein solution are considered interfering substances in a protein assay if they artificially suppress the response, enhance the response, or cause elevated background by an arbitrarily chosen degree (e.g., 10% compared to control). Additional components can include reducing agents, chelators, crowding agents, and protease inhibitors.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
There are several criteria that should be considered, including compatibility with the sample type and components, assay range and required sample volume, protein-to-protein uniformity, speed and convenience for the number of samples to be tested, and the availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Unfortunately, no protein assay method exists that is either perfectly specific to proteins (i.e., not affected by any nonprotein components) or uniformly sensitive to all protein types (i.e., not affected by differences in protein composition). Therefore, successful use of protein assays involves selecting the method that is most compatible with the samples to be analyzed, choosing an appropriate assay standard, and understanding and controlling the particular assumptions and limitations that remain. The objective is to select a method that requires the least manipulation or pre-treatment of the samples to accommodate substances that interfere with the assay. Each method has its particular advantages and disadvantages. Because no one reagent can be considered the ideal or best protein assay method for all circumstances, most researchers have more than one type of protein assay available in their laboratories.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Unfortunately, no protein assay method exists that isn’t affected by any non-protein component or uniformly sensitive to all protein types. One must select an appropriate assay method based on compatibility with the sample type or one that requires the least manipulation of the sample to accommodate the assay. Most researchers will have more than one type of assay available in their laboratories.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We offer several types of protein assays including the: BCA Assay, BCA-RAC (Reducing Agent Compatible) Assay, Micro BCA Assay, 660 nm Protein Assay, Pierce Bradford Plus Protein Assay Kit, Pierce Bradford Protein Assay Kit, Modified Lowry Assay, colorimetric and fluorometric Peptide Assays, CBQCA kit, EZQ kit, Quant-iT kits, NanoOrange, and the Qubit kits.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Pierce Bradford Plus Protein Assay Kit has all the advantages of the regular Pierce Bradford Protein Assay plus the added benefit of having a more linear standard curve. The added linearity of the standard curve translates to higher accuracy in the protein assay.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The assay has a detection range of 1 µg/mL to 1,500 µg/mL of protein. The protein of interest must have a molecular weight of at least 3,000 daltons to be detected by the assay. For peptides smaller than this, we recommend the Fluoraldehyde o-Phthaldialdehyde Reagent Solution (OPA) (Cat. No. 26025). This will fluorescently detect amino groups on small peptides.
Other peptide quantitation assays we offer are Thermo Scientific Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275) and Thermo Scientific Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The Pierce Bradford Protein Assay Kit is incompatible with high detergent concentrations. This can be overcome by removing the detergents from the protein solution. The Thermo Scientific Detergent Removal Resin can be used to remove the detergents from protein solutions. Other methods for removing detergents include size exclusion, dialysis, or protein precipitation.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The Pierce Bradford Protein Assay has several advantages over the traditional Bradford Method. The first is that there is no reagent preparation required; there is no dilution or filtering necessary and can be used right out of the bottle. Secondly, the reaction is very fast and results can be achieved as quickly as in 30 seconds.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The assay is compatible with many agents that are incompatible in other assays. The Pierce Bradford Protein Assay has been shown to be compatible with amine-containing buffers, reducing agents, chaotropic agents, organic solvents, sugars, antimicrobial agents, DNA, protease inhibitors, low concentrations of metal chelators, and metals.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
The assay is based on the absorbance shift from 465 nm to 595 nm that occurs when Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution. The mechanism of action of the Pierce Bradford Protein Assay is that the anionic form of the dye complexes with proteins, resulting in a color change from reddish brown to blue. The dye is assumed to bind via electrostatic attraction of the dye's sulfonic groups. The dye has been shown to interact chiefly with arginine residues and weakly with histidine, lysine, tyrosine, tryptophan and phenylalanine residues. The reaction reaches a stable end-point so absorbance does not drift over time.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Please check the Protein Assay Compatibility Table in this Tech Tip (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf) to determine which protein assay would be most effective with your sample type.
Find additional tips, troubleshooting help, and resources within our Cell Lysis and Fractionation Support Center.