I prepared sample for SDS PAGE from serum in reducing conditions using Laemmli buffer and heating it for 10 min at 95 degrees. But due to aggregation I couldnot load the sample on SDS PAGE anymore, how to prevent this ?
If your samples are becoming lumpy, The cause could be DNA rather than protein aggregation. Try Sonication or DNAse treatment.
I work with serum and I haven't experienced any problem because of aggregation of the sample. I think that the problem could be the pseparation of the serum from the whole blood.
You have to make sure the serum will be without traces of red blood cells. To do that you have to wait at least 30 minutes so that the clotting process will complete and after that you can procede with the centrifugation to obtain the serum.
Richa: I don't know how much serum and Laemmli buffer (ie, their ratio) you were using in your treatment. My guess is on top of Nancy and Mitesh's discussions, that serum is a biofluid with exceptionally high concentration of proteins. In my own experience using serum for WB, I only used as little as 0.4 microL per lane (diluted in PBS first and then mixed with Laemmli or directly in Laemmli) prior to heating. This way, I had never seen any aggregation.
First of all, are you sure that your samples are serum of blood not plasma of blood?
Secondly, protein concentration of such samples are very high (more 60 mg/ml).
I diluted the testing samples before heating till concentration 1mg/ml.
Hi,
if you have potassium ions in your sample this will lead to aggregation with SDS (K-SDS has low solubility). It is soluble in high temp. but precipitates when cooling down. Either you dialyse your sample to remove potassium before adding the SDS-buffer or you try to pipet the sample while it's still hot and hope it doesn't precipitate in your pipet tip before loading it into the gel pocket. Sounds funny, but believe me I have done this many times (mostly because of laziness or lack of time for dialysis or both), the best way is having the heating block at ~70°C next to the gel while loading. Btw, the same happens with samples containing guanidinium.
Hope that helps.
The problem is in the serum preparation. It seems that there is an hemolysis in your samples.
@Nancy Duarte, you have mentioned that you are frequently working with blood serum. Would it be possible for you to share your protocoll regarding the serum preparation prior SDS page/western blot. I would greatly appreciated that. Thank you.
- Badges
- Science method