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Problem with control samples in cell IC


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#1 elpollodiablo

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Posted 24 September 2010 - 05:28 AM

Hi,

I'm trying to stain for Troponin I in a cardiac cell line. I've done this by blocking in 4% normal goat serum, use primary antibody from origene raised in rabbit and used anti-rabbit secondary raised in goat and coupled to a fluorophor. Problem was that I got a lot of staining in my control with cells that certainly do not express my target protein. However, no staining in the negative where I omitted primary antibody. I did some optimization, blocked with 10% instead of 4% NGS and used different concentrations of primary antibody (1:500, 1:1000, 1:5000).

Now, I don't think the secondary antibody is the problem as there is no staining when I omit the primary antibody, but perhaps it has something to do with blocking. Does anyone have have suggestions how to solve this problem? I thought about perhaps changing the composition of the blocking buffer to include BSA or milk.

Thank you in advance!

#2 rkay447

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Posted 24 September 2010 - 07:16 AM

I would recommend you try to block in 5% filtered BSA, use the highest dilution that gives you acceptable staining in your troponin positive cell line, do a second block in 5% goat serum and then the secondary. Wash your cells very well...you can use PBST (PBS + 0.1-0.5% Tween) to help get rid of background. I have an antibody that I have to wash overnight to get rid of major background. The best test of your primary is to see if you can get your hands on the peptide that the antibody was generated against. You pre-incubate the antibody with this peptide and then try to stain the cells. Any staining that remains after pre-absorbing the antibody with the peptide is non-specific. Also, I just looked this antibody up on the company website and they do offer the protein but it is expensive. I also noticed that they recommend this antibody for western at 1:1,000,000 (!!! I've never seen an antibody dilution this high!!!) and for IHC 1:250,500. I would guess that you are using way too much antibody and this is giving you outrageous background staining. You really need to do a dilution test to determine what the highest dilution you can use that stains your positive cells. Then use this dilution on your negative control. I would start at a 1:100,000.

#3 elpollodiablo

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Posted 24 September 2010 - 07:20 AM

Thank you for your reply. I'll certainly look into your remarks in details. About the dilutions recommended by the company, I thought it meant 1:250 OR 500. If indeed it means 1:250,500 I'm way off! I'll check with the company. Thanks again.

#4 rkay447

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Posted 24 September 2010 - 10:22 AM

Thank you for your reply. I'll certainly look into your remarks in details. About the dilutions recommended by the company, I thought it meant 1:250 OR 500. If indeed it means 1:250,500 I'm way off! I'll check with the company. Thanks again.

You know, I thought that might be as well but the western blot definitely says 1:1,000,000 which is why I think the IHC means 1:250,500. But the most important is to find the highest dilution possible that still works in the positive control. This way you are reducing the potential for background staining in the negative control.




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