Question (14518) | Anti-SNAP25 antibody (ab5666)

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Question

BATCH NUMBER 137071 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM no signal by Western blot of mouse trigeminal ganglia protein extracts or immunohistochemistry on mouse brain, using recommended primary antibody dilutions SAMPLE Western blot: mouse trigeminal ganglia total protein extract PRIMARY ANTIBODY SNAP 25 (1:2000) in 1% albumin in 1xPBS containing 0.005% Tween-20, incubation: 2 hours, wash with 1xPBS containing 0.05% Tween-20 DETECTION METHOD Pierce SuperSignal West Dura Extended duration substrate POSITIVE AND NEGATIVE CONTROLS USED I am writing to request information about an appropriate positive control. ANTIBODY STORAGE CONDITIONS 20 ul aliquots stored at -20 C SAMPLE PREPARATION lysis buffer: 1% triton x-100, 2 mM EDTA in 1xPBS Calbiochem protease inhibitor cocktail 3 min boil AMOUNT OF PROTEIN LOADED 100 ug ELECTROPHORESIS/GEL CONDITIONS sample buffer: reducing conditions, specifically, contains Tris-HCl, SDS, glycerol, and B-mercaptoethanol gel: 18% Tris-HCl TRANSFER AND BLOCKING CONDITIONS blocking: 1% albumin in 1xPBS containing 0.005% Tween-20 SECONDARY ANTIBODY Pierce Goat anti-Rabbit HRP-conjugated (1:1500) x 2 hour wash with 1xPBS containing 0.005% Tween-20 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? longer antibody incubation time

Answer

I'm very sorry to hear you are experiencing problems with ab5666 in both WB and IHC, this antibody has not been tested on sections (only in cell culture) and without a detailed IHC protocol it is hard to determine what the problem may be in that application. We have tested the antibody in WB on rat and mouse brain lysate and therefore with the following recommendations I hope this will improve you signal: - use a stronger lysis buffer to extract the protein (e.g RIPA buffer which contains a strong detergent) -incubate the membrane in blocking buffer (5%BSA) in TBST (Tris buffer with Tween 0.1%) for 1 hr then -incubate the membrane in antibody at 1:1000 overnight (diluted in TBST Tween 0.1%) at 4C Please rinse in TBST too and check that your secondary antibody works well with other primary antibodies. These steps will promote the antibody binding and enhance the signal. If you would like me to look at your IHC protocol I enclose a link to the IHC protocol questionnaire, if you put it to my attention I will look at this as soon as I receive it: https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=5666&mode=questionaire Please let me know if you need further assistance,

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