Need help with my SDS-PAGE gels - (Sep/22/2017 )

Hello. 

I cast my gels using the BioRad recipe. I wash the wells with water multiple times before loading my samples. Yet, I always get these smear like lines. 

Are those due to the sample collection being less than optimal or the wells need better cleaning?

it could be due to "skins" in the wells. they can form if the plates are warped or the spacers and comb don't match exactly (the comb may wear).

 

however, based on lanes 1 and 6, i suspect that the problem is the samples themselves (unless there is no sample in 1 and 6). is there kcl in the sample? it looks like a precipitate formed.

mdfenko on Sun Sep 24 19:11:10 2017 said:

it could be due to "skins" in the wells. they can form if the plates are warped or the spacers and comb don't match exactly (the comb may wear).
 
however, based on lanes 1 and 6, i suspect that the problem is the samples themselves (unless there is no sample in 1 and 6). is there kcl in the sample? it looks like a precipitate formed.

Lanes 1 and 6 are ladder lanes.
I use lysis buffer for collection and sonicate theb spin for an hour at 14K xg.

what's in your lysis buffer?

 

detergents like triton can displace sds and distort the run.

mdfenko on Mon Sep 25 20:23:32 2017 said:

what's in your lysis buffer?

 

detergents like triton can displace sds and distort the run.

150 mM sodium chloride 0.1% NP-40 (Triton X-100 can be substituted for NP-40) 50 mM Tris pH 8.0.

The way I do it, I use 200uL with 0.1% NP-40, sonicate then add 300uL lysis buffer without NP-40 and sonicate again. All have Phosphatase and protease inhibitors and DTT

both np-40 and triton x-100 can displace sds. also, the nacl can also distort the run.

Okay. How can I get around this?

you can do a buffer exchange to reduce salt and eliminate detergents in the sample prior to adding sds sample buffer. you an use spot dialysis if you want to prepare just enough for the gel sample.

 

you can dilute the sample with salt and detergent-free buffer if the protein is concentrated enough.

Do you mind linking me to protocols to do do any of the suggested steps?

for spot dialysis, float a piece of dialysis membrane on the buffer. then put a drop of sample on the membrane. no agitation.