I am trying to probe for a protein by western blot. I have homogeneized paw skin in lysis buffer which was then centrifuged for 50 mins at 13000 rpm. The supernatant was then taken and made into samples suitable to run on the gel. My westerns is very messy with high up bands that I believe are due to sticky matrix proteins that the antibodies just loves to stick to. I was going to try precipitation with ethanol (9x ethanol, overnight, centrifuge, resuspend pellet). Does anyone think this will help?
You can use centricon centrifugal filter. This is a simple way to avoid unwanted protein in western. I used the same filter to discard higher MW protein from supernatant.
You need to purchase according to you protein size, suppose your protein size is 50 kD you can you 30 kD filter to elute out smaller size protein and if your protein size is smaller you can do vice-versa followed by concentrating your sample.
I think this way you can avoid unwanted contamination.
Ever consider using MMP enzyme? And perhaps combine it with the solution from Banshi above?
Hi Samantha, if you are using good antibodies, they should not be binding to anything besides you antigen.
If you are getting clear bands on SDS-PAGE and non-specific binding on westerns, then the problem might be due to blocking buffer that you are using for the western.
Do you include serum in your blocking buffer?
Samantha, without seeing your results it is difficult to diagnose your problem. I would be interested in seeing your protocol to see if we can't identify the problem that way.
Some good things to note: use SDS, insure complete homogenization of your tissue, pellet any insoluble material and transfer cleared lysate to new tube.
Banshi has given a good suggestion, but first make certain that your lysate preparation protocol is okay.
Best of luck.
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