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Western Blot: Unequal Sample Migration - (Aug/01/2014 )

Hello-

 

I'm currently running a western blot with the intent of working out ideal staining conditions.  Hence, I have the same 3 proteins repeating on one large gel, and will cut the nitrocellulose and expose them to different Ab concentrations and incubation times.  However, I just loaded my gel and noticed that my liver sample is "smiling" within its lane (see attached image).  Does anyone know what typically causes this phenomenon?

 

Details:

 

1) ~50mg skeletal muscle or liver was homogenized with a bead mill.  RIPA w/ protease inhibitors was used.

 

2) Performed a BCA to quantify total protein.  Performed in duplicates.

 

3) Normalized a portion of each protein sample to a concentration of 2ug/uL using the same RIPA w/ protease cocktail.

 

4) Added 100uL 2x Laemelli to 100uL of each sample.  Final concentration protein = 1ug/uL

 

5) Loaded 20uL (20ug) sample per well.  Began run - 150V.  Smiling of Liver sample was observed.

 

I've been using this chamber, gel and buffer system for months - haven't had this issue.  However, I previously was loading E.coli lysates that were in 8M Urea.  The fact that the adjacent muscle sample is running fine makes me think it's sample specific.

 

Would protein overloading cause this?  I'll look over my math, but thought posting here might be helpful.

Thanks in advance.

 

 

 


Attached Image

-djvan-

And after a quick check I think I've found my problem.  When transferring my data (manually) from the BCA output into an excel spreedsheet, I made a typo.  41.00 ug/mL is not quite the same as 14.00 ug/mL.   blink.png

 

Looks like I actually loaded 57ug protein.  I assume this is what's slowing migration down?  What do you think my western's will turn out like?  Just thinking I might not continue with these while I troubleshoot....

-djvan-

with the increased protein you will also have increased lipid.

-mdfenko-

with the increased protein you will also have increased lipid.

What factors slow down the migration?  Increased concentration I imagine has to do with it?  How does lipid content affect migration (you've hinted that it hinders migration, but what's the science behind it?  Interactions with polyacrylimide?)

-djvan-

lipids affect migration. i think it may block pores. also, the detergents necessary to solubilize the lipid will have an effect (maybe the free detergent in the samples with less lipid (ie- the non-liver samples) are migrating faster due to the detergent present from the ripa?).

 

lipids, detergents, salts, buffers, etc. all have an effect on migration. we always try to equalize these factors between samples although some really can't be (eg lipids).

-mdfenko-