Author Topic: Secondary Antibody Diluent  (Read 7982 times)

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Offline funk.106

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Secondary Antibody Diluent
« on: January 18, 2011, 09:53:56 AM »
Hi, so I've got a basic question about western blotting. For the most part, I block my western blot in 4% nonfat milk, and then dilute both antibodies in that blocking buffer and have success. I recently purchased a new antibody, however, for which the data sheet says not to use milk as my blocking agent--to use BSA instead. So I tried to perform a blot--blocked with 5% BSA in TBST and then diluted both primary (Rabbit) and secondary (Goat anti-rabbit) antibodies in 1% BSA-TBST (no sodium azide in either dilution)--and I got nothing. I'm pretty confident that the actual blot worked (markers look good), but I am wondering if I was right to dilute my antibodies in 1% BSA...particularly the secondary? What do you dilute your antibodies in in such a situation? Is there anything I need to be particularly careful about?

Secondary Antibody Diluent
« on: January 18, 2011, 09:53:56 AM »

Offline MT Scientist

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Re: Secondary Antibody Diluent
« Reply #1 on: January 18, 2011, 10:06:17 AM »
Diluting the secondary in BSA will cause no harm.  I would suggest staining your membrane with Coomassie to see if your samples/bands of interest tranferred (MW band transfer is no guarantee).  If your bands are there, I would look at your primary and secondary to make sure nothing was omitted or overlooked.

BSA is better if phosphoproteins are involved.  Milk contains phosphoproteins that will cause background.

Offline funk.106

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Re: Secondary Antibody Diluent
« Reply #2 on: January 18, 2011, 10:21:00 AM »
I know that the protein of interest transferred because I can probe for the unmodified protein successfully. I think either the antibody isn't working or I'm using it wrong.

Offline MT Scientist

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Re: Secondary Antibody Diluent
« Reply #3 on: January 18, 2011, 10:48:53 AM »
Need more information...

Offline funk.106

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Re: Secondary Antibody Diluent
« Reply #4 on: January 18, 2011, 04:05:41 PM »
yes, I realize mostly I'm just going to have to troubleshoot on my own. We bought a new ECL kit, which may help boost a weak signal, which I noticed on a previous blot. If that doesn't fix the problem, I'm going to try some dot blots to optimize conditions. I was just curious what people dilute their antibodies in when they can't use milk.

Offline MT Scientist

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Re: Secondary Antibody Diluent
« Reply #5 on: January 18, 2011, 04:49:19 PM »
If you were a bit more specific (i.e. post your protocol), perhaps more ideas would be forthcoming.  As it stands now, all a person can do with the limited information you provided is guess....

Re: Secondary Antibody Diluent
« Reply #5 on: January 18, 2011, 04:49:19 PM »