std for bradford assay - (Feb/11/2009 )
How u usualy prepare std for the bradford assay when your lysis buffer is RIPA?
I don't know but my std curve with ripa abd BSA is funny/
Sure, Bradford very sensitive to any detergents presence. If you sample prepared in buffer with a detergent (like RIPA) you should use other methods to determine total protein concentration. I would recommend BCA assay (like one from Pierce # 23225)
The detergent is throwing everything way off. I always dilute the bradford reagent with water and then just add 1ul of the protein sample to 1mL of the reagent. My standard curve with BSA is always gorgeous and I seem to get accurate quantification of the samples. If you really want to be accurate, you can do one sample of bradford with 1ul of Ripa alone. This is the "background" absorbency the buffer and detergent is contributing to the protein sample readings and you subtract this from the sample readings. However, the buffer is being diluted 1:1000 so it really is minor.
I will try the less amount of lysis buffer and let you know what will happen.
rkay447 on Feb 12 2009, 10:43 AM said:
rkay447's results notwithstanding, using bsa for the standard with the bradford method gives erroneous results with many (if not most) proteins. it has been found that bgg (bovine gamma globulin) is a better standard to use for many proteins when using the bradford protein assay.
we found that to be the case for all of the proteins that we routinely isolate (we checked the results with several different methods using both bsa and bgg as standard). you may want to confirm this for your protein of interest, as well.
of course, the best standard to use for any protein assay is the purified protein with which you are working.
I have heard this myself and never paid much attention to it. I was told that BSA binds up approximately twice as much bradford reagent than the average protein so your calculated sample concentration from a BSA standard curve is much lower than reality. I've always meant to do the standard curve with IgG and compare to BSA standard done at the same time. Just seems like I'm in too much of a hurry and just want to know the sample. Also, I usually do bradfords with a curve when trying to get relative concentration amoung many unknown samples and always get great results. If I just need a rough idea of the concentration of a single sample I just take the od595 of the one sample and calculate according to a curve I've previously done. Not the most accurate by any stretch of the imagination but it gets me close enough to set up IPs. I have a labmate who just multiplies the od595 by 15 and surprizingly it gets pretty close to the concentrations I get when I do a full out bradford with curve.
if it works for you then, by all means, continue (not that you need my permission).
we required more accuracy (we were developing new purification methods and needed to give accurate recovery data, plus we're a bit anal).