Keeping proteins from washing off the slide

[TheWalkingGlitch]

Keeping proteins from washing off the slide

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Post by TheWalkingGlitch » Fri Oct 28, 2011 8:10 am

I have protein patterns on a bare slide, and I want them to be fluorescent so I incubate them with a primary antibody then a secondary labeled antibody, but the wash afterward is causing the pattern to come off. I've tried washing with PBS as well as DI water. I have also tried various coatings for the slide, such as glutaraldehyde, silane derivative and agarose. The only acceptable patterning results are with bare slide. But I'm trying to keep the patterns from washing off. I would appreciate any suggestions anyone has in order to make the protein stick better. Maybe something to mix into the protein solution that won't block the antibody, or something to make a thin coating on the slide? Any websites or papers would be welcomed. If there must be a film, thinner film is better, ideally nothing between the protein and the slide. Thanks in advance

[daniel.kurz]

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Post by daniel.kurz » Sun Oct 30, 2011 2:34 am

Try a dot of clear nail polish.

[TheWalkingGlitch]

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Post by TheWalkingGlitch » Sun Oct 30, 2011 2:51 am

Thanks for your response, but I wouldn't want to cover the protein and keep the antibodies from binding.

[daniel.kurz]

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Post by daniel.kurz » Sun Oct 30, 2011 8:18 pm

Sorry I misread the first time. I thought you were using well slides. Why do you have the protein on a slide and not run into a membrane? Are you trying to see this protein in its native form or under a microscope?

[daniel.kurz]

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Post by daniel.kurz » Sun Oct 30, 2011 8:21 pm

I've never thought about trying to bind an antibody to a protein that is fixed to a slide. The only way that I can think of binding the protein would be to use magnetism and a tag on the protein or otherwise charge the slide. Its an odd sort of experiment.

[TheWalkingGlitch]

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Post by TheWalkingGlitch » Sun Oct 30, 2011 9:01 pm

OK thanks for your comments. I am trying to see patterns of fluorescence under the fluorescent microscope. I was actually thinking that my actual washing technique might be the problem, however this issue only recently started happening. I've tried washing by squirting with the pipette as well as dunking the slide briefly, then should I leave it to dry at room temperature, in the fridge, or in the incubator? I've also tried a gentle compressed air stream to dry the slide. Just wondering if there's a standard protocol for washing.