For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
LOT NUMBER XXXXX ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Multiple bands or non-specific bands. SAMPLE Cell lysates of: A20 cell line (mouse), Devernelle cell line (human), mouse lymphocytes. PRIMARY ANTIBODY Anti-SH2D1B antibody (ab95270) abcam. Diluted 1/250 in PBS-Tween 0,1%. (Previously tritated) Incubated during 2 hours (when we tried 1 hour we observed NO bands)at room temperature in agitation. Washed three times during 10 minutes with PBS-Tween 0,1% at room temperature. DETECTION METHOD ECLPlus. POSITIVE AND NEGATIVE CONTROLS USED Positive: Jurkat cell lysate Negative: mouse serum lysate ANTIBODY STORAGE CONDITIONS 4°C, NOT sub-aliquoted. SAMPLE PREPARATION Cells are lysed with the following lysing buffer heated at 100°C prior mixing with the cells (we use a ratio of 8 million cells for 300 uL of lysing buffer): -SDS 10% -Tris-Cl 1M pH 7,4 -PMSF 100 mM -Na3VO4 0,1 M -Calbiochem protease inhibitor cocktail AMOUNT OF PROTEIN LOADED 20 ug and 40 ug (in both cases we obtain the same multiple bands) ELECTROPHORESIS/GEL CONDITIONS 12% polyacrilamide gel. Run at 50V during 30 min, then 100V for 1 hour. Using Electrophoresis buffer (Tris 25mM, Glycine 0,2M, SDS 0,1%) TRANSFER AND BLOCKING CONDITIONS Transferred during 2 hours at 4°C, using Transfer Buffer (Tris 25mM, Glycine 0,2M, Methanol 20%. Bloking agent: PBS-Tween 0,1% + 3% BSA, blocked over night at 4°C. SECONDARY ANTIBODY DakoCytomation Polyclonal Goat Anti-Mouse Immunoglobulins/HRP Code No./ Code/ Code-Nr. P 0447 Diluted 1/5000 with PBS-Tween 0,1%. Incubated for 1 hour at room temperature in agitation and in darkness. Wash step: washed three times for 10 minutes at room temperature with PBS-Tween 0,1%. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 30 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
Asked on Jan 05 2012
Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody. Reviewing this case, I would like to offer some suggestions to help optimise the results from ab67417. I would also appreciate if you could send me an image of any of the failed blots, in order to better understand why this antibody did not perform as stated on the datasheet. - Could you please confirm the order number? It will help us to track the -Attending your protocol, both Mouse and Human samples have been used, however, this antibody has only been characterised using human cell lines, and we don’t have any evidence of its suitability for Mouse samples. -I would recommend using less concentrated antibody solutions, as 1/500 and 1/1000, and let the antibody incubate at 4 C overnight. -For the sample preparation, we suggest using RIPA buffer, which has a higher denaturing level. The recipe to prepare it can be found under the link: https://www.abcam.com/index.html?pageconfig=resource&rid=11414 It is also important to use stronger reducing conditions, supplementing the lysis buffer with DTT or mercaptoethanol, and heating the sample at 95C for 5 minutes. -It would be interesting to try different blocking conditions, as, 5% of non fat milk in TBST, for 1-2 hours at RT. -Regarding the antibody storing conditions, if no cycles of freezing/thawing are done, the antibodies are viable for up to one year at 4C, so in this particular case, if the antibody was stored for 3 months before being used, there shouldn’t be any degradation issue. I hope the above recommendations may help you, if you still experience problems please do not hesitate to contact me with an image of the blot if possible. I would also appreciate if you could also provide details of your order (purchase order number, date of purchase, shipping address/purchasing agent information) in order to dismiss any shipping issue, as well as to monitor the concrete lot complaints. Please do not hesitate to contact us if you need any more advice or information.
Answered on Jan 05 2012