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Author Topic: IF for multiple antigens under differing incubation conditions  (Read 5109 times)

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Offline robertg

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IF for multiple antigens under differing incubation conditions
« on: October 28, 2008, 12:41:04 PM »
I wish to perform double staining for 2 antigens. The first requires incubation at 37oC, whereas the second works best at 4oC overnight. I am worried that I am losing the first signal during the second incubation period...
Is it possible to fix the first antibody before continuing to the second one (without losing affinity for the secondary antibody)?

Many thanks,
Robert

IF for multiple antigens under differing incubation conditions
« on: October 28, 2008, 12:41:04 PM »

Offline richard03

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Re: IF for multiple antigens under differing incubation conditions
« Reply #1 on: October 28, 2008, 11:00:44 PM »
Have you tried both antibodies at room temperature? Majority of antibodies should work well at room temperature incubation for 1 hour.

Offline robertg

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Re: IF for multiple antigens under differing incubation conditions
« Reply #2 on: October 29, 2008, 08:30:01 AM »
the antibody that requires 37oC does not work at RT. the other antibody works ok at RT.
Robert

Offline Cardio

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Re: IF for multiple antigens under differing incubation conditions
« Reply #3 on: November 10, 2008, 01:48:51 PM »
You just answered your own question then.  Do both at 37.

Offline robertg

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Re: IF for multiple antigens under differing incubation conditions
« Reply #4 on: November 11, 2008, 06:27:35 AM »
Unfortunately, the other antibody doesn't work so well at 37oC...

Robert

Offline richard03

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Re: IF for multiple antigens under differing incubation conditions
« Reply #5 on: November 11, 2008, 07:03:38 AM »
I suggest you use sequential approach. Do the one that works best at 4 C FIRST. Then perform another one that works best at 37 C.

Offline Cardio

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Re: IF for multiple antigens under differing incubation conditions
« Reply #6 on: November 11, 2008, 11:10:35 AM »
Richard has the most logical way of doing it. The first one should be overnight at 40 then the second at 37 for whatever time.

These really sound like low affinity ab's. You might want to look for better quality ab's.

Offline robertg

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Re: IF for multiple antigens under differing incubation conditions
« Reply #7 on: November 11, 2008, 02:13:28 PM »
Thanks very much.

Offline ImmunoNYC

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Re: IF for multiple antigens under differing incubation conditions
« Reply #8 on: November 14, 2008, 01:46:42 PM »
Does the one that works at 37 deg C really not work at RT (25 deg C)? I find that incredibly surprising .........

Re: IF for multiple antigens under differing incubation conditions
« Reply #8 on: November 14, 2008, 01:46:42 PM »