Question (16264) | Anti-RASSF1a antibody [3F3] (ab23950)

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Question

BATCH NUMBER 133336 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM I got different staining pattern when the cells were fixed at methanol or 4% paraformaldehyde. When the cells were fixed by methanol, the staining located at cytoplasm. When the cells were fixed by 4% paraformaldehyde, the staining was accumulated at the nucleus. I also didn't see the localization described in the datasheet at different cell cycle status. I wonder if Abcam has tried the condition of IF of this antibody? SAMPLE Flesh Hela cell on coated slide-chamber PRIMARY ANTIBODY Abcam RASSF1A antibody (1:200), overnight DETECTION METHOD IF ANTIBODY STORAGE CONDITIONS stock: -20 working aliquot: 4 FIXATION OF SAMPLE Methanol/paraformaldehyde ANTIGEN RETRIEVAL NO PERMEABILIZATION STEP PBS with 0.1% Triton X-100 BLOCKING CONDITIONS 10% goat serum, 10 min room temp SECONDARY ANTIBODY Zymed FITC-goat anti-mouse IgG, 1:100, 30 min HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

Answer

I have received feedback today from the laboratory that tested ab23950 and was informed that they have used A431 cells fixed with 4% PFA, methanol has not been tested. Cells were labeled with ab23950 and detection was achieved using a biotinylated secondary antibody and Texas-red conjugated streptavidin. In many cases, the results of staining showed speckled pattern or weak staining in cytoplasm but we couldn’t observe microtubule stained with this antibody. In mitotic cell, we could observe the mitotic spindles stained with this antibody and so thought it was kinetochore stained specifically. The laboratory confirmed that the rest of your protocol looks very similar to yours and believes 4% PFA fixing is most suitable for this antibody. Please find on the datasheet the image of the staining, I hope the above information and image will help,

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