Zero absorbance by Bradford assay for Dialyzed protein - (Sep/02/2013 )
Hi,
Since I have purified my protein of the interest using denaturing method, I had to get rid of urea before mice injection. I tried to dialyze my protein against reducing concentrations of urea in PBS. The dialysis has been done successfully as no aggregation happened. But when checking the protein concentration using Bradford assay, not only color change was not happened by adding the protein sample to Bradford reagent but also the concentration reported by the spectrophotometer was zero! However, after running the dialyzed protein on the SDS-PAGE the band of protein was visible! There were no detergent or other Bradford interferer in the protein elution buffer or in PBS. Could anyone help me with this problem?
Thanks
Since you see the right protein on the gel but not in the bradford assay, either (i) the bradford assay was done incorrectly (did you do it side by side with a positive control, like BSA?), (ii) the protein is insensitive to bradford (some proteins don't work well with bradford...have you done it before on this protein and know that it should give a signal?), or (iii) you're not adding enough volume of your protein solution to the assay (use at least 1ug in a ml of reagent).
Alternatively, try the BCA assay. Works great.
Even better- since your protein is pure, why not just check absorbance at 280 nm? Use the calculated extinction coefficient for your protein sequence and you should get a good idea of how much protein you have.
labtastic on Wed Sep 4 20:36:21 2013 said:
Since you see the right protein on the gel but not in the bradford assay, either (i) the bradford assay was done incorrectly (did you do it side by side with a positive control, like BSA?), (ii) the protein is insensitive to bradford (some proteins don't work well with bradford...have you done it before on this protein and know that it should give a signal?), or (iii) you're not adding enough volume of your protein solution to the assay (use at least 1ug in a ml of reagent).
Alternatively, try the BCA assay. Works great.
Even better- since your protein is pure, why not just check absorbance at 280 nm? Use the calculated extinction coefficient for your protein sequence and you should get a good idea of how much protein you have.
1) I performed the assay using standards (BSA) and the blank. 2)I have done the bradford assay for this protein before dialysis and it was ok. 3) I added 20 microliter of the protein sample regarding the protocol. But after dialysis, I could not check the absorbance using bradford assay
Why can't you read absorbance at 280nm to determine protein concentration?
labtastic on Sun Sep 8 18:28:43 2013 said:
Why can't you read absorbance at 280nm to determine protein concentration?
I did it.but the result was zero again! I think sth happens to my protein after dialysis that interferes with my protein but I can't find it!
Yesterday,I repeated the dialysis for some other sample of the same protein, this time although the result of the bradford was not zero,it was very low. but the band of the protein was sharp again on SDS-PAGE!
What is the amino acid content of your protein?
Thanks for your reply.Sorry for my delay in responding. Here is the sequence of my protein:
MADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAPAAAAEEKTEFDVVLADGGANKINVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAKVELKETKVEWFGTVRARLGYTATERLMVYGTGGLAYGKVKSAFNLGDDASALHTWSDKTKAGWTLGAGAEYAINNNWTLKSEYLYTDLGKRNLVDVDNSFLESKVNFHTVRVGLNYKF
try reading at 215nm. this will read peptide bonds instead of ring structures (280nm).
are you sure the bands you see in the gel are your protein and not an artifact?
mdfenko on Tue Oct 15 12:57:54 2013 said:
try reading at 215nm. this will read peptide bonds instead of ring structures (280nm).
are you sure the bands you see in the gel are your protein and not an artifact?
Thanks for your reply. Yes,I'm sure.It is single sharp band in the expected size! While reading at 215nm, should i add any reagent to my protein sample or it is like reading at 280nm by nonodrop?
you read it the same way as you read at 280nm. blank against whatever buffer the protein is in then read the sample.