Hi all,
I have been trying to run a BN-PAGE now a few times and have been getting smears down the lanes with no clear bands (No smears in negative control lanes) (Blot attached for visual reference). My protein of interest is an integral membrane protein fused with eGFP (protein shown to still be functional with addition of eGFP). The native extraction buffer (EB) used is 1x NativePAGE sample buffer (ThermoFisher), 1x protease cocktail inhibitor, 1%DDM and prepared fresh. 30 minute incubation of of crude tissue sample in EB on ice.
Crude sample is then undergone immunoprecipitation using anti-GFP magnetic beads and eluted in a way to preserve native structure.
0.25% coomassie G-250 is added to samples and then 10ul is applied to a Bis-tris 3%-12% pre-cast gel cassette lanes. Ran gel using 120v for 2 hours, using appropriate running and cathode buffers . Blotted at 4 degrees using Tris, glycine and methanol transfer buffer onto a PVDF membrane. Blocked using 5% milk powder in TBST buffer. Primary anti-GFP antibody applied (1:10000) 4 degrees overnight, washed using TBST, secondary antibody, HRP conjugate (1:5000), 1hr30 room temp, washed using TBST. ECL Select WB regent used to visualise blot.
My initial thoughts were that I was loading far to much protein. I subsequently diluted my samples 2x and 4x but smears still were present. After doing a bit of research, this may be an issue with my protein being ineffectively solubilised (in which integral membrane protein can be). In the future, new detergents may be trailed, along with higher concentrations of DDM in hope to achieve clearer bands. Could this also be an issue in my antibody concentration, with such uniform smears and sample dilution, I thought unlikely.
Sorry for the ramble, let me know what you think? Any advice would be amazing.
I would start with a dot blot or an Eliza to know if the protein if interest is still on your sample
Dear Felix
why are you running a BNPAGE insted of a SDSPAGE? If you have to see the result of a native purification protocol run small quantity in SDSPAGE the result will be much clear.
In my experience integral membrane proteins i.g. with 3 spanning domains (viral proteins) sometime can precipitated after heating and remain in the well without run at all. In SDS_PAGE proteins run because SDS give them the charge to move in the electric field. In BNPAGE proteins run only with own charge in a running buffer with only 0,1% SDS. In my opinion your protein is there because the smear suggest it, but the majority is in the wells, it does not run properly . Try to use a stronger loading buffer such as 25mM Tris-HCl pH 6.8, 3%SDS , 5%BMe, 10%glycerol.
You can chek the expression by fluorescent microscopy , if your protein is expressed it will carry the GFP at the membrane. The result should be clear also by FM
best wishes
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