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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunohistochemistry In Situ Hybridization forum
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blocking endogenous biotin
Posted by: ivison (IP Hidden, New member, 1)
Date: June 22, 2005 01:40PM
Hi,
I am trying to stain PFA-fixed HeLa/Caco cells with a biotinylated primary Ab and detecting with Streptavidin-FITC. I can not differentiate transfected and nontransfected cells and it may be because of high amounts of endogenous biotin. Any tricks to get around this? I do not understand the blocking with Strepavidin: if I do it before adding primary Ab, then my biotinylated Abs will bind to background, if I do it after, it will bind my Abs. Washing with a biotin-containing buffer after adding both Abs may help but who is to say that the endogenous Streptavidin-biotin interactions are going to be weaker then my biotinylated Ab-Streptavidin-FITC bond? Am doing a double staining, has anyone tried using SUBSEQUENT anti-mouse and anti-rabbit secondary Abs? Thanks for any tips...
Re: blocking endogenous biotin
Posted by: annem (IP Hidden, Unregistered user, )
Date: June 30, 2005 12:47AM
Hi!
You should try to block twice, first with streptavidin, after washing with biotin. Then you must wash again and then you may continue with treatment with your biotinylated Ab. I found nice protocol from Pierce - block endogenous biotin. Have fun! :)
Re: blocking endogenous biotin
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 30, 2005 03:16PM
ivision, I hope this helps. The streptavidin block binds all endogenous biotin (vitamin H is found in all living cells); this will prevent a high background that may interfere with probe detection or purification. The subsquent incubation with biotin will block all remaining streptavidin-biotin binding sites with free biotin. This will prevent streptavidin from binding your probe (antibody) used in the experiment. Block samples as usual with a blocker containing protein (BSA, non-fat milk, etc.) diluted in TBS. Cover sample with streptavidin (0.1mg/ml) diluted in wash buffer (TBS with/without 1% BSA and with/without 0.05% Tween 20) and incubate for 15 minutes at room temperature. I usually leave the BSA out, due in part to the expensive, and do not have any trouble. Wash 3x for at least 10 minutes. Add biotin (0.5mg/ml) and incubate for 45-60 minutes at room temperature. Wash and then continue as usual with your biotinylated antibody (biotin-labelled probe). Please let me know if you have any other questions.
Re: blocking endogenous biotin
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 30, 2005 03:21PM
If you do it the way I suggested, your antibodies will not bind to the background nor will they be bound by streptavidin. You aren't really doing double staining just by using this protocol. You can use subsquent anti-mouse and anti-rabbit secondary Abs as long as you no there is no cross reaction.
Re: blocking endogenous biotin
Posted by: salva (IP Hidden, Unregistered user, )
Date: July 21, 2005 03:22AM
Hello,
I am studying for master thesis. I want double immunohistochemistry. I am working rat kidney tissue. Although I tried some methods, I didn't block endogenous biotin. This methods; Peroxidase method; %30 Hydrogen peroxide 3 ml, Distilled water 97 ml. (or Methanol). 2 egg whites diluted to 200 mls with distilled water for this purpose. Are there any practical methods? I pleasure to help at this issue. Thank you. Emine Salva
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