Question (25295) | Anti-Calcium channel L type DHPR alpha 2 subunit/CACNA2D1 antibody [20A] (ab2864)

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Question

LOT NUMBER GR1538-2 ORDER NUMBER 950823 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE mouse cell PRIMARY ANTIBODY Concentration or dilution 1:500 and 1:1000 Diluent buffer 5% skim milk Incubation time 3 hour Incubation temperature: RT Wash Buffer TBST Number of washes 4 times x 3min DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED - ANTIBODY STORAGE CONDITIONS -20C SAMPLE PREPARATION Lysis buffer RIPA Protease inhibitors: yes Phosphatase inhibitors no Reducing agent yes Boiling for ≥5 min? yes, 5min or 10min AMOUNT OF PROTEIN LOADED 30ug/lane ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS Type of membrane Nitrocellulose Protein transfer verified yes, ponceau S staining Blocking agent and concentration 5% skim milk Blocking time 1 hour Blocking temperature RT SECONDARY ANTIBODY Species: goat Isotype: igG Reacts against: rabbit Concentration or dilution 1:2000 Diluent buffer 5% skim milk Incubation time 1 hour Incubation temperature: RT Fluorochrome or enzyme conjugate: HRP Wash Buffer TBST Number of washes 4 times x 3min HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Antibody dilution factor & blocking buffer

Answer

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this product. Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem. - Could you please specify the cell type of the mouse cells used for these experiments? - Total cell lysates or membrane preparations have been run on the gel? I look forward to hearing from you soon.

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