I was seeking suggestions regarding a problem I am having with my western blot.
During the stacking phase my samples have tails behind which i guess is leading to smeared bands when i observe the membrane after transfer.
I have tried changing every reagent including the gel buffers, sample buffers running buffers and the acrylamide solution itself. I load 40ug of protein and have also tried loading lower.
Need suggestions please.
Thanks in advance.
Hi Mohd Najib Mostafa . I think the problem is more with your samples. Samples that have higher salt concentrations run slower and are more diffuse. Samples that have a lot of aggregated proteins look like they have many dye fronts.
I suggest centrifuging your samples at 14,000 rpm for 5 min to pellet the aggregated proteins and lower the salt concentrations.
Dear all, it seems your samples have a very broad molecular weight distribution, their relative mobilities are very distinct. Fractionation is the solution. My Regards
Dear Steingrimur Stefansson I tried centrifuging my samples, and collecting the supernatents before making the sample with loading buffer. but it was still the same.
Hi Mohd Najib Mostafa . Try running just the sample buffer itself (without the proteins) mixed with the SDS loading buffer.
For example K+ , arginine and guanidinium in a buffer can mess up SDS PAGE.
Hi Mohd
I think it's about the composition of the sample. Look at line 2 and 12 (left to right), they have a yellow color that supposed an acid pH in the environment. High salt concentration and viscosity cause distortion in the dye front that can be removed by desalting in column or dyalisis. TCA - acetone precipitation can be an alternative. Centrifugation or filtration remove insoluble material but no salts!
Dear all, One thing I noticed was that when preparing the sample with loading buffer after quantification, if i use Distill water as diluent to make the total volume and not the RIPA buffer I used previously for lysis the sample ran much better. So am thinking how my lysis buffer can affect this. I use 20mM Tris pH 7.5, 150mM NaCl, 1% NP-40 1% Na deoxycholate, 1mM EDTA and Protease + phosphatase inhibitors cocktails.
Dear Oscar Otero Alfaro lanes 2 and 12 look a bit yellow as they were the markers.
Steingrimur Stefansson I tried only the sample buffer and they had no tails.
Mohd Najib Mostafa . Is there no SDS in your RIPA lysis buffer? The high concentrations of NP40 and deoxycholate in your lysis bufferr could be preventing SDS to interact with the proteins and alter their mobility on the gel.
Dear Steingrimur Stefansson , I use SDS in my sample Buffer, should I be using SDS in Lysis buffer too?
Hi Mohd Najib Mostafa . Using SDS in RIPA buffers is quite common. Here is a thread on RG.
https://www.researchgate.net/post/Best_recipe_for_Ripa_buffer
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