Serial Dilution Bsa Standard Curve
I was trying to quantify protein of whole brain tissue homogenate. The buffer used in the sample is RIPA buffer. So, when i did the serial dilution for BSA standard, i used RIPA as diluent. But i found that the standard curve look so abnormal n weird. As i know that, detergent can affect the Bradford assay.
But then, what can i use to dilute my BSA to draw my standard curve? And wat can i can do with the sampe which is already mix with the RIPA buffer? Is there any other idea to help out this? Because i only have bradford reagant.Anyone knows bout this? Can someone help? I routinely use RIPA to lyse cells for various applications.
I agree with mdfenko. Perform a microassay and just dilute your samples/standards in water (or PBS), but add a volume of RIPA equal to the sample volume to each of your standards.For example.I use a total of 1 ml for the Microassay.
(800 µl water or PBS + 200 µl Bio-Rad Protein Assay Reagent)If performing a Bradford on 5 µl of sample then add 5 µl of RIPA to the Blank or Standard +? Μl volume Standard + remaining volume up to 800 µl with Water. Then add Protein Reagent, mix, incubate, and read.I hope this isn't too confusing! I found this thread by searching and am glad to be reminded that detergent throws off the Bradford assay.My standard curve looked fine, as it was BSA diluted in TE. I never tried to make a standard curve in any detergent-containing buffer.However, based on this standard curve, I was told by the Bradford assay that all my samples were within 2-fold of each other. But then the bands on the gel (GAPDH) looked like there could be more like a 20-fold difference between the highest and lowest concentration.The Bradford assay did rank the samples in almost the right order from highest to lowest concentration - but the relative quantities were all off.
I could also detect this by looking at the concentrations the Bradford told me - and then doing the 2-fold or 1.5-fold dilutions which should bring them all to the same concentration - only to find that it hardly had any effect on the relative differences.Is this the sort of thing expected from samples lysed/diluted in RIPA buffer, which is only about 1.2% detergents? (No loading buffer/sample buffer had been added). I found this thread by searching and am glad to be reminded that detergent throws off the Bradford assay.My standard curve looked fine, as it was BSA diluted in TE. I never tried to make a standard curve in any detergent-containing buffer.However, based on this standard curve, I was told by the Bradford assay that all my samples were within 2-fold of each other. But then the bands on the gel (GAPDH) looked like there could be more like a 20-fold difference between the highest and lowest concentration.The Bradford assay did rank the samples in almost the right order from highest to lowest concentration - but the relative quantities were all off. I could also detect this by looking at the concentrations the Bradford told me - and then doing the 2-fold or 1.5-fold dilutions which should bring them all to the same concentration - only to find that it hardly had any effect on the relative differences.Is this the sort of thing expected from samples lysed/diluted in RIPA buffer, which is only about 1.2% detergents?
(No loading buffer/sample buffer had been added)you should always either add buffer to your standards or, at least, run buffer blanks to determine the offset caused by the buffer. I routinely use RIPA to lyse cells for various applications.
I agree with mdfenko. Perform a microassay and just dilute your samples/standards in water (or PBS), but add a volume of RIPA equal to the sample volume to each of your standards.For example.I use a total of 1 ml for the Microassay. (800 µl water or PBS + 200 µl Bio-Rad Protein Assay Reagent)If performing a Bradford on 5 µl of sample then add 5 µl of RIPA to the Blank or Standard +? Μl volume Standard + remaining volume up to 800 µl with Water. Then add Protein Reagent, mix, incubate, and read.I hope this isn't too confusing!Hi all,I am new to the forum and I had a question regarding the Bradford assay. I use a recipe for lysis buffer to lyse insect cells and I want to measure the protein concentration by bradford microassay.
As such, I set up my bradford similarly to what Roo mentioned but using a final volume of 500uli.e. For the standard curve, BSA @ 1mg/ml(1ul,2ul,3ul.) + lysis buffer (5ul) + water (394ul, 393ul, 392ul.) + Bradford reagent(100ul). For my sample I add 5ul(already in lysis buffer) in 395ul water + 200ul Bradford reagentWhen I read my sample concentration off the standard curve, what is the concentration units - is it ug/ml or ug/ul?Also, what dilution factor do I need to multiply by to get the actual sample concentrations?If using a 96 well plate, is the linear range the same as if using cuvettes?Forgive the simplistic questions but I am getting confused trying to figure this out!Thanx.
The is a sensitive technique for determining the concentration of a protein in a liquid sample. When performing ELISAs, three different data outputs can be generated:. Qualitative. An ELISA can be used to determine whether the protein of interest is found within a particular sample by comparing the sample to a blank well or a sample that does not contain the target protein. Semi-quantitative. Samples can be compared to one another to determine the relative amount of protein within each sample, as the intensity of signal is proportional to the concentration of the target protein.
Quantitative. The amount of target protein within a sample can be precisely determined by setting up a standard curve of known target protein concentrations using purified antigen.The preparation of the ELISA standard curve is critical in accurate sample quantification. This is a brief guide to preparing a standard curve and using the standard curve to quantify protein amounts. Preparing the Standard Curve. Choice of standardThe standard curve should be prepared using purified protein.
Some companies sell purified proteins that are suitable with ELISA assays. Purchased ELISA kits should contain a standard.If purified protein is not available, then recombinant protein can be semi-purified in the lab and the concentration determined by HPLC. Standard curve rangeTypically, an ELISA measures protein concentrations in the range of 0.1-1 fmole or 0.01-0.1 ng, however this is dependent on the antibody-antigen interaction. Therefore, a classic standard curve ranges from 0-1000 pg/ml, although some can go as high as 3000 pg/ml if the samples to be measured are concentrated. Reconstitute the standardIf the standard is supplied lyophilized, then it must be reconstituted prior to first use. To reconstitute the standard:. Mix each dilution well but avoid creating bubbles or foaming.Prepare a new standard curve for every experiment and each plateA standard curve run at different times will not have the same OD values for each dilution.
Serial Dilution Bsa Standard Curve Test
This is due to operator differences and slight differences in pipetting, incubation times and temperature. Therefore, a new standard curve should be prepared for every experiment. Likewise, if analyzing more than one plate in an experiment, each plate should contain its own standard curve. Using the Standard Curve to Quantify Samples. Once the intensity of each well has been measured on the plate reader, calculate the average absorbance values for each duplicate/triplicate sample.
Then generate a standard curve by graphing the mean absorbance for each sample (x-axis) vs. The standard concentration (y-axis). Typically, a standard curve will have a sigmoidal shape in which the higher concentrations of standard dilutions will reach a plateau in absorbance.Find the portion of the curve that is linear and draw the best-fit trendine for the data (many ELISA plate readers have built-in programs for generating and analyzing standard curves or you can use Microsoft Excel, or similar graphing software). Use the computer software to generate the equation of the line (i.e. Y=mx + b; m= slope of the line and b= y intercept) and the R2 value.
Bradford Bsa Standard Curve
The R2 value is an indication of how closely the data fit the trendline. An R2 value of 1 is perfect.Calculate the concentration of each sample by using the average of the duplicate/triplicate samples for x in the equation.If the concentration of the sample exceeds the highest point of the curve or does not lie within the linear range of the curve, then dilute the sample prior to measurement. If a diluted sample is used, remembered to multiply by the dilution factor to obtain the final value.Photo courtesy of.