Anti-BMX antibody (ab59360)

LOT NUMBER XXXXX ORDER NUMBER XXXXX DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE - Species: Human and mouse - What’s cell line or tissue: human lung cancer cell line-A549、 mouse lung cancer cell line-LLC1、TC1 and primary lung tissue cells. - Cell extract or Nuclear extract: Cell extract - Purified protein or Recombinant protein: Total cell lysate PRIMARY ANTIBODY - Species: Rabbit - Reacts against: reacted to human BMX and predicted to react to mouse - At what dilution(s) have you tested this antibody: 1:500 and 1:1000 - What dilution buffer was used: 5% BSA in TBST. - Incubation time: 4°C overnight - Incubation temperature: 4°C overnight - What washing steps were done: To wash the protein transferred membrane with TBST three times. DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Yes, the positive control cell line-A549. ANTIBODY STORAGE CONDITIONS 1002399 SAMPLE PREPARATION - What lysis buffer was used: RIPA lysis buffer containing several protease and inhibitors - What protease inhibitors were used: Roche coattail package、NaVPO4 and NAF - What loading buffer was used: 2-ME containing sample buffer - Phosphatase inhibitors: purchased from sigma - Did you heat the samples: temperature and time: 95 °C, 10min AMOUNT OF PROTEIN LOADED 50ug ELECTROPHORESIS/GEL CONDITIONS - Reducing or non reducing gel: SDS containing reducing gel - Reducing agent: SDS - Gel percentage : 12% TRANSFER AND BLOCKING CONDITIONS - Transfer conditions: (Type of membrane, Protein transfer verified): The membrane was PVDF and the transfer condition followed in molecular cloning” - Buffer: TBST - Blocking agent: milk, BSA, serum, what percentage: 5% BSA in TBST. - Incubation time: 1hr - Incubation temperature: Room temperature SECONDARY ANTIBODY - Species: Goat - Reacts against: reacted to rabbit - At what dilution(s) have you tested this antibody: 1:4000 - Incubation time: Room temperature for 1hr - Wash steps: To wash the protein transferred membrane with TBST three times. - Fluorochrome or enzyme conjugate: HRP - Do you know whether the problems you are experiencing come from the secondary? no 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? concentration of primary antibody

ANSWER:

Thank you for contacting us and taking the time to send the questionnaire and the image. I am sorry to hear you have been experiencing problems with one of our antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints. This antibody has never been characterised in mouse samples, and therefore we cannot guarantee it will work in this species, it is nevertheless predicted to react with mouse due to the high percentage of homology between the sequences. In any case, the positive control (human A549) should show a band at approximately 78KDa and it is clearly not the case of the image sent. After reviewing the protocol it looks perfect to me. The only suggestion I would make is increasing the protein rate in your samples, and for that, using a cytoplasmic lysate instead of a total cell lysate. Running a no primary control could be useful to dismiss any secondary failure. I would also encourage you to contact us next time you need to test an antibody from us in an untested species/application. By doing it, we can offer you a testing discount for a primary antibody purchase if you test the antibody and submit your results to our website as an Abreview. To find out more about our Abreview system, please visit: http://www.abcam.com/abreviews. Anyway, I am more than happy to help you, and, as your order was placed within the last 4 months I can still send you a replacement from the same antibody of from a different one against the same target, please have a look at the alternatives: http://www.abcam.com/BMX-antibody-Y396-ab32153.html http://www.abcam.com/BMX-antibody-ab24515.html Otherwise, I can offer you a refund or a credit note for a future purchase in compensation. Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.