Weird BSA standard curve for Bradford assay - (Jun/12/2009 )
hai! 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? thanks!
you can try diluting your samples and ripa diluted bsa with water and performing a micro-bradford determination.
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!
Hmm,what are the importance and the uses of BSA Standard curves?
Any links to view?
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)
MDavies on Nov 9 2009, 03:08 AM said:
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.
Roo on Tue Jul 7 19:39:47 2009 said:
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 500ul
i.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 reagent
When 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
are you really adding 100ul reagent to the standards and 200ul to the sample or is it just a typo?
assuming it is a typo, you can evaluate the standard curve as mass (rather than concentration), hence, you have 1ug, 2ug, 3ug,...
you can determine concentration afterward (Xug/5ul).
mdfenko on Fri Feb 4 20:25:34 2011 said:
are you really adding 100ul reagent to the standards and 200ul to the sample or is it just a typo?
assuming it is a typo, you can evaluate the standard curve as mass (rather than concentration), hence, you have 1ug, 2ug, 3ug,...
you can determine concentration afterward (Xug/5ul).
Thanks. Yes sorry that ws a typo. it should be 100ul.
what i have been doing and what seems to make sense is to dilute your lysis buffer to say 1:50 and use that as blank and diluent. Then dilute your samples to 1:50 as well with water and results should be comparable.
i do this even with a detergent compatible assay.