Problem with SDS-PAGE gel - (Aug/11/2013 )

Dear all, 

              I am having problem with running proteins samples on SDS-PAGE. I don't know the cause but I can show you the problem. I am uploading the pictures of the problem. I read earlier about how to troubleshoot SDS-PAGE. I have tried new Sample buffer and lower the APS and TEMED conc. for stacking gel I have also tried not to screw the plates tightly but nothing worked. First picture is just at the start of the SDS-PAGE and second one of the same PAGE after a while. 

I would highly appreciate if someone could explain the problem to me. Its really messing up my experiments.

Thank you 

What is the voltage you run your SDS-PAGE? Do you use cooling water circulator?

Adrian K on Sun Aug 11 17:05:30 2013 said:

What is the voltage you run your SDS-PAGE? Do you use cooling water circulator?

I run at 120V(constant). I never used cooling water circulator in fact we never do in the lab except during transfers. I am having this problem for last 3 PAGEs I have done, not before. Thanks

hi,

i had sth similar recently and then i realized that instead for a stock buffer 1.5M Tris pH 8.8 i was using 1M Tris pH8.8.

just be sure for your stock solutions, your running buffer to be fresh, check the mA instead the Voltage, boil your samples @95C for 5-10min and spin them before loading onto the gel.

origami on Sun Aug 11 19:38:28 2013 said:

hi,

 

i had sth similar recently and then i realized that instead for a stock buffer 1.5M Tris pH 8.8 i was using 1M Tris pH8.8.

 

just be sure for your stock solutions, your running buffer to be fresh, check the mA instead the Voltage, boil your samples @95C for 5-10min and spin them before loading onto the gel.

We only have 1.5M Tris pH8.8 so thats perhaps is not the problem. We prepare our running buffere fresh. I will check again for these things. Did change to 1.5M solved the problem? did you change anything else?

thanks

Do you degas your gel solu. before pouring? You should do that as air bubbles do create problems. Looks like your gel has not set properly. Was there a minute leak?

Shefali Desai on Mon Aug 12 09:20:43 2013 said:

Do you degas your gel solu. before pouring? You should do that as air bubbles do create problems. Looks like your gel has not set properly. Was there a minute leak?

I don't degas, In fact I never had to. This problem is recent. I believe it was set properly cause the remaining gel in falcon was hard as a rock. There is always a bit of leakage which is normal. I still dont know what is the problem.

Is there someone who could look at the pictures and tell me what could be wrong?

bio_film on Sun Aug 11 19:58:26 2013 said:

 

origami on Sun Aug 11 19:38:28 2013 said:

hi,

 

i had sth similar recently and then i realized that instead for a stock buffer 1.5M Tris pH 8.8 i was using 1M Tris pH8.8.

 

just be sure for your stock solutions, your running buffer to be fresh, check the mA instead the Voltage, boil your samples @95C for 5-10min and spin them before loading onto the gel.

We only have 1.5M Tris pH8.8 so thats perhaps is not the problem. We prepare our running buffere fresh. I will check again for these things. Did change to 1.5M solved the problem? did you change anything else?

 

thanks

 

yeap it did change...that period i was doing co sedimentation assays and i had to be really careful with my gels because i wanted to quantify the bands...which means that i had to have perfect made gels....and i dodnt know why i didnt pay attention the concentration of the stock buffer...i took it as a granded until one colleugue noticed this.

are these the same samples as you've run in the past?

if not, is there salt with the sample?

are any of the solutions you are using newly prepared (just before your gels went south)?

how old is the sds?