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Author Topic: What dilution for IgG negative controls and blocking peptide  (Read 2512 times)

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Offline njcragg

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What dilution for IgG negative controls and blocking peptide
« on: November 20, 2003, 04:54:03 AM »
Please could anyone tell me what dilution to use for a rabbit IgG negative control?  Also I would like to know what dilution / concentration of blocking peptide is optimal?  How long do you have to incubate the blocking peptide and the primary antibody together before incubation on the section?

Thanks in advance,

Nic
ic

What dilution for IgG negative controls and blocking peptide
« on: November 20, 2003, 04:54:03 AM »

Offline ghostdog

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What dilution for IgG negative controls and blocking peptide
« Reply #1 on: November 20, 2003, 10:31:14 AM »
It depends, if it is just a normal negative control it should be at the same dilution as the antibody your using to get a positive result, if you are using  a blocking peptide you should use it at the highest dilution at which you seen positive staining in your work up, as far as i know it is usually left at 4 degrees over night !!

hope this helps

Offline Jason

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Peptide Blocking
« Reply #2 on: November 24, 2003, 08:02:22 PM »
If you would like to use peptide blocking as a test for specificity, you need to pre-incubate the antibody to it's working concentration and combine with the peptide (1-20 g/ml concentration).  Make sure that there is more peptide than antibody. Pre-incubation should be at least 2 hours @RT, overnight @ 4C is best. Once pre-incubated, apply the "blocked" antibody solution to the slide as you would a normal antibody, and proceed regularly. This peptide block should eliminate the positive result that you obtain when applying the antibody (without peptide).  An additional negative control would be to incubate an additional slide with a non-specific peptide at the same concentration; this should not eliminate the positive result.

This works for me,
Jason LeCocq

Peptide Blocking
« Reply #2 on: November 24, 2003, 08:02:22 PM »