Hello
I use the Bradford method to measure the amount of protein expressed by E.coli BL.21. My problem is that despite the R² = 0.9814, the average absorption read at 6 and 21 hours after adding the IPTG that I put in the equation is a negative number. In fact, it shows the amount of protein after 21 hours in an environment such as negative LB, while it has an acceptable band on SDS-PAGE gel.
Thank you for your help.
Thanks
Dear Fatemeh
sorry but are you performing bradford quantification on the purified protein or are you would like to extrapolate your protein amount performing the Bradford assay the total E.coli protein and comparing induced with non induced E.coli?
In the first case, negative results could be due to high dilution of the sample (under assay sensitivity) or not correct baseline correction
In the second case, i think that this approach is very difficult, first of all because, in the most cases the expressed protein represent only a small % of total E.coli protein and the variation also if is positive is not so high, and also because IPTG induction slow down E.coli growth and if fron one side the protein amout is increase because your target protein is expressed from the other side the other protein are less and it may happen that total protein is reduced and you observe negative values.
If your protein carryng a tag (eg Hid,tag) i suggest you to perform a small purification trial and quantify the protein eluted by bradford.
Ciao
good luck
Manuele
Hello
I was measuring the total E.coli protein. Yes, my protein has His-Tag, so I purify it first and then measure its concentration with Bradford assay.
Thank you very much for your explanation and suggestion.
Dear Fatemeh
at the folloqing link you can find some tips to perform small scale imac
https://www.researchgate.net/project/Optimizing-new-protocolls-for-recombinant-protein-production/update/5b8a5dac3843b006753741ff
good luck
Manuele
What are you using as blank? If that has a higher absorbance than your sample, you get a (meaningles) negative result.
Plot your absorbance vs concentration data and make sure they are on a straight line. High r in regression on a computer doesn't ensure this.
Also, Bradford is very sensitive to interference by detergents, high salt, polyols and a lot of other tings.
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