I have a protein extract that is not purified, I ran it through SDS-PAGE and observed bands of 150-200 kDa and other smaller ones of between 60 and 8 kDa. A colleague ran my samples through FPLC with a 6HR superose column, however in the FPLC protein profile I see peaks of less than 20 kDa, but not larger ones. What could have happened to the larger proteins?
A possibility is that the proteins were in the form of large aggregates that were not able to penetrate the Superose column, but could be resolved on SDS-PAGE because they were denatured and unfolded by SDS.
Here are a couple of questions;
What were the absorbance wavelengths of the choice for the FPLC application?
Which injection volume and concentration of the proteins were used for FPLC?
Do you have information about the total protein concentration and AA sequences?
What was the injection solution composition?
Is your sample fully solubilized in the mobile phase for the SEC application?
Did you observe and backpressure increment after SEC-FPLC injections?
Are your results reproducible with SEC? Is there any fluctuation?
Have you ever tried to denature your proteins first by using concentrated urea and injecting them to 6HR superose after proper dilution?
You may add DTT to reduce your proteins and also to see differences.
Have ever tried to eliminate the matrix components? I mean isolating the proteome from other endogenous compounds by ultrafiltration or precipitation?
I'm guessing you are doing reduced/denatured SDS-PAGE, so your protein bands are not necessarily going to match what your large proteins doing natively on the column, so the larger proteins are potentially eluting in the VOID volume.
If you share your chromatogram, buffer conditions of extract and GF buffer, and the SDS-PAGE we'll be in a better place to help interpret your results.
Dear Daniela Dalzotto
as Adam B Shapiro suggest, the SDS-page is denaturing (proteins run as monomer) while in the SDS-page will run as aggregate in case the form is oligomeric, is it possible that those large aggregates that were not able to penetrate the Superose column even if it is quite stange since if i'm not wrong the superose 6 is designed to speratate very high MW complexes (5MDA? i'm right?)
i think that since the sensitivity of SDS-page is higher than the sensitivity of the UV, is it also possible in case that you can have a problem of detection due to the loading of low sample amount.
which volume sample concentration did you load in the SEC coloumn?
best
Manuele
I also had the same problem, so the best things you can do before subjecting it to FPLC.
1) Try to dilute the samples 1:5, 1:10 or 1.20.
2) You Can Add DTT.
centrifuges the samples properly.
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