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BATCH NUMBER 154754 ORDER NUMBER unknown DESCRIPTION OF THE PROBLEM Wrong band size. ~75kDa, not 170kDa. Similar results with ab5638 and ab5644. SAMPLE Rat heart tissue lysate. PRIMARY ANTIBODY listed in inquiry DETECTION METHOD Pierce ECL or Pierce Supersignal. POSITIVE AND NEGATIVE CONTROLS USED PC-3 lysate ANTIBODY STORAGE CONDITIONS Aliquotted and stored at -20. SAMPLE PREPARATION Tissue lysis buffer: 50mM HEPES pH7.5 150mM NaCl 1.5mM MgCl2 1mM EGTA 10% glycerol 1% Triton X-100, plus protease inhibitor tablets, plus 10mM NaF and 1mM NaVO4. All done in cold room/on ice. Lysates kept at -80 when not in use. Typical western setup, sample buffer, boil, 7.5% gel. Transferred to PVDF 100V 1 hour, checked transfer with Ponceu S. Blocking/dilution buffer is TBS 2 or 5% BSA fraction V. AMOUNT OF PROTEIN LOADED Usually around 100ug. ELECTROPHORESIS/GEL CONDITIONS reducing & see above TRANSFER AND BLOCKING CONDITIONS see above SECONDARY ANTIBODY Zymed specific to each of the Abcam primaries' species. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 15 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Homogenized with and without phosphatase inhibitors, transfer time, blocking/dilution buffers, incubation temp, incubation volume, antibody concentrations, washings, development time
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ANSWER: |
Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have been in touch with the originators of ab5638, ab5644 and ab14017 with regards the band sizes that you have detected. I have received the following comments. The mentioned that the most likely explanation for the bands that you have been detecting and the mass of protein that you have been loading is that the the cell preparation that you have been using does not contain any EGFR phosphorylated at these 2 specific sites (ab5638 and ab5644). When the correct target does not exist (or exists in concentrations too low for the antibody to detect), the antibody will bind to either a lower affinity phospho. protein or to the most concentrated protein in the lysate: Neither bindings are specific. The originator also recommended that you try titrating the extracts themselves and the secondary antibody to determine whether the target protein is indeed the protein detected by the antibody or a non-specific band detected by the secondary antiserum. I would like to recommend that you stimulate cells using EGF to induce phosphorylation as shown in the western blot on the datasheet of ab5638 where EGF induced phosphorylation of EGFR. I hope this information helps, please do not hesitate to contact us if you need any more advice or information. |
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