The Western Blot Gods are Angry with Me

I am doing Western blots today, and none of them are working.

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For those of you who do not know what a Western blot is, a Western blot is a technique to detect proteins in a sample of cell lysates. First the proteins are purified. Then the protein in the sample are suspended in loading buffer that contains a detergent called SDS and boiled. This denatures the proteins and coats them in a negative charge (SDS is negative). The proteins are then separated by weight in an electric field (bigger proteins move more slowly through the gel) and transferred to a sticky membrane. Finally the Western blot part begins where antibodies are used to detect the proteins on the membrane.

A little piece of trivia: the Western blot is so named because of a bad pun. There is an analogous procedure for detecting DNA called a Southern blot, named after Edwin Southern. Since this is for protein they called it a "Western" blot. There is another technique for detecting RNA called a "Northern" blot. I know...hardy har har...molecular biologists just kill me.

So my Westerns aren't working for reasons I do not entirely understand. Perhaps I am not appeasing the Western blot gods. Perhaps, the Western blot gods demand a sacrificial goat. Quick, I need a goat, a young priest, and an old priest.

A lab mate of mine has suggested that the problem might be in the animals the antibodies came from. Antibodies (polyclonal ones) are made by injecting a peptide into a rabbit or a goat or something. My lab mate has suggested that if I send carrots to said goat that it will be happier and make better antibodies. Somehow I doubt it.

UPDATE: Success! I have conquered above described Western blot. And the crowd goes wild!!!! I am at present doing a touchdown dance in my lab (suck on that image). Science -- I own you!

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First, is that your gel or blot in the pic, or just a random pic you found online? What's not working about them? You're not getting any signal at all? Or is it not specific? Are you using primary and secondary antibodies, or is your primary antibody tagged for detection? And what detection method are you using? Have you stained the gel to make sure that the protein's there and in sufficient quantity? Everything seem OK with whatever you're using to blot?

Just some preliminary troubleshooting...

Thank you for the offers of help but I think I got it now.

I think the issue was that I had to strip this blot (doing a phospho-protein to total protein ratio) and all of my antibodies for total protein are not very good. I was starting with an IP of the total protein, and I didn't want to reblot with that antibody because the background would be ridiculous. But, I think I got it now. I just upped the concentration of my bad antibodies and made sure to was excessively to get out the nonspecific binding.

That blot is not the current one. It is rather one of my earlier travesties as an example.

I am loving that I can get all this troubleshooting help though. Thanks guys.

Dude, have you tried the TrueBlot secondary conjugate from eBioscience for these here IP/Westerns? Their secondary only recognizes native IgG (i.e., your primary) and not the bungload of pulldown antibody that is now nicely denatured into heavy and light chains on your blot. The only added goat-f*#k, however, is that you can't use protein A/G beads for the immunoprecipitation - you've got to use anti-species IgG beads instead.

But, hell, between Shelley and Professor Smith, I'd send 'em my extracts and a few bottles of wine if I'm having trouble (no disrespect intended, just admiration for your troubleshooting skills and willingness to help.).

The blot that you show looks like an incomplete strip - I can't think of any reason you'd get those big ol' blank spots unless the post-strip primary had nothing to bind to since the pre-strip primary was bogarting the antigen.

mmm i am having the same problems with my Western blots, it's nice to see i'm not alone! wish me luck!

From what I know blots look like this when too much ECL is added. Some how the ECL reagent reacts with your proteins and can completely denature them. So you end up getting these spot where you have not signal. If you pay close attention the white spots with no signal get bigger and bigger with time. ie your first <1 min exposed film will usually have smaller white spots. And all your subsequent films will have bigger white spots with no signal. Sometimes I am able to fix this problem by washing off the ECL reagent and then putting less ECL reagent for a shorter about of time (2mins instead of 5min incubation in ECL) before exposing the film to the blot. anyways my experience.

Dear Dr Young
I am having the same problems with my Western blots, I mean the white bands!!
what does these bands mean? it means high expression of our protein or it merge because too much ECL is added?
I'll be really appreciated if you guide me.

My Email address: shabani_khadijeh@yahoo.com

Best,

By khadijeh shabani (not verified) on 25 Feb 2010 #permalink

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