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-   -   IF detection of a tiny cytoplasmic protein: fixation method? (http://www.molecularstation.com/forum/confocal-microscopy-imaging-techniques/73848-if-detection-tiny-cytoplasmic-protein-fixation-method.html)

Lizzzie 09-16-2010 04:29 PM

IF detection of a tiny cytoplasmic protein: fixation method?
 
Hi everyone
One of my colleagues wants to detect a 3 kDa cytoplasmic protein (actually, a 20aa peptid fused with His and V5 tags) by immunofluorescence (he transfects it then wants to check his transfection).
I was wondering what kind of fixation/permeablilzation method he should use to retain such a tiny protein... I guess ethanol/methanol would be too harsh, so maybe formaldehyde or buffered formalin, with triton permeabilization?
Any recommendation would be great!
Thanks :)
Liz

Warthaug 09-17-2010 09:31 PM

Re: IF detection of a tiny cytoplasmic protein: fixation method?
 
Methanol doesn't really fix cells - it sucks out all the water and removed membranes. Your protein may be lost during this, or it may be succesfully trapped.

Paraformaldehyde works quite will - it'll chemically cross link your protein to surrounding ones, holding it in place. My usual protocol is 30min of 4% PFA at 4C (you can go longer if you desire). PFA should be made in sailne, PBS or HBSS - DO NOT make it up in protein/peptide/amino-acid containing buffers, nor buffers containing amide groups, as the PFA will react with those buffers. Dissolving PFA is a real bitch - best bet is to buy pre-made 16% PFA. Simply dilute 1:4 in PBS and use.

After fixation you'll need to permeabilize. I generally do this in PBS + 0.1% triton X-100 + 5% donkey or goat serum. Add to sample and incubate 1hr at room temp.

To stain with antibody, dilute your primary in the same buffer used to permeabilize the cell at the desired dilution (generally 1:100 to 1:2000). Incubate at 4C for 4 hours to overnight. If there is too much non-specific staining, try using a shorter incubation period at room temp.

Wash 3-5X with PBS, 15min/wash.

Add secondary antibody in permeabilization buffer, at desired dilution (generally 1/2 the concentration of the primary). Incubate 1-2hrs at room temp.

Was 3-5X with PBS, 15min/wash

Mount and image.

============
A hint to save antibody. If using small coverslips (18mm circulars, 25mm squares) you can use the "hanging drop" method to save a lot of antibody. Dilute the antibody in a final volume of 100ul/coverslip. Put the drop of antibody on a clean piece of parafilm, and invert the coverslip onto the dot. If done carefully, the coverslip should float on the antibody solution and not have any air bubbles. You can also do this with larger coverslips, but you'll need a larger volume of antibody (150ul for 25x50mm coverslips, etc). If you cover this with a lid, and keep it in the fridge, you can easily stain overnight without it drying out.

Hope that all helps.

Bryan


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