I am doing western blots for UCP1 in mouse BAT and I have not had problems with previous blots, however, recently all my blots have had the same streaking in the bands. Below are pictures of one of my previous blots and one that I did today. I am not sure as to why this is occurring. I use 12.5ug of protein in each well, and use 5%skim milk blocking. I haven't changed my protocol in anyway either.
Hi Erik,
Firstly, for these 2 blots, did you use the same protein lysate?
Slight differences in protein extraction protocols (e.g. lysis buffer, sonication time) may yield different qualities of protein within your lysate.
I believe the amount of protein being loaded is not an issue at all, but I did recall getting smears like this more commonly when dealing with protein lysates obtained from tissue samples and not from cells. Considering that the smears appear on all your samples on your newer set of experiments, I think the problem lies more in your gel or protein preparation and you can rule out differences in protein expression.
Some things you may consider:
- Did you make a stacking gel that has enough length? Sometimes, separation of your protein may require more length of stacking gel.
- Have you checked the quality of your buffers (i.e. any turbidity or fungal contamination?)
- Did you use protease inhibitors when preparing the protein samples?
- Did you homogenize your samples thoroughly and ensure that your extracts are clear?
Best of luck!
Each lane is whole tissue lysate from a different mouse. I was thinking it could be a problem with the length Of the stacking gel, But I was also wondering if it could be a problem with the polymerization of my gels?
Also, when I transfer my protein, I cut the membranes after so I can blot for alpha tubulin additionally. The UCP1 protein in the pictures is about 32kDa. I don’t see any problem in the alpha tubulin blots when I do this. I did this with all 4 blots in the pictures.
Hi Erik,
was it a fresh preparation? I have also suffered this problem with old samples. Change the PI and follow the other recommendations.
Fast electrophoresis causes streaking.
What Deepak is completely true, if you had collected the protein samples at the same time and ran the gels at 2 different times, there is a greater chance of getting smears like that, especially for proteins obtained from tissue samples. Given what you said about the alpha tubulin, perhaps you can try and run the UPC1 protein separately if you have adequate amount of samples left? Sometimes control proteins appear too abundantly and may yield carryover effects due to degradation.
Did you boil your samples in sample buffer? Although that is the standard procedure you may get better results if you heat to 60 °C only, for 10-15 min.
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