ANSWER: |
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Thank you for your reply.
I have processed your request to receive the 2 free of charge replacements and below is the testing discount code for ab93942.
Please let me know if there is anything else I can help you with.
DISCOUNT CODE:**********
Expiration date: *********
I am very pleased to hear you would like to accept our offer and test ab93942 in rat tissue. This code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview forrat tissue and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.
Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.
Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.
The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount. |
For the Sci Sup,
Customer is getting no bands with 3 different antibodies (ab93942, ab32351 and ab2000). Please, find below the questionnaire filled out by the customer. Let me know if you need further information.
Abcam WB Questionnaire
1) Abcam product code:
ab93942 (Rabbit polyclonal to DNM1L)
ab32351 (Rabbit monoclonal [E87] to Caspase 3)
ab2000 (Rabbit polyclonal to APAF1)
2) Lot number:
3) Antibody storage conditions (temperature/reconstitution etc):
All antibodies at -20ºC.
4) Description of the problem (high background, wrong band size, more bands, no band etc.):
We can’t see bands for all antibodies.
5) Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.):
Wistar rats’ brain (hippocampus, cortex and striatum).
6) Sample preparation (Buffer/Protease inhibitors/Heating sample etc.):
For the first time, the extraction was made with RIPA buffer with protease inhibitor.
After, we tried a different extraction (for mitochondrial and cytosolic proteins → 10mM Tris-HCl pH 8, 1mM EDTA pH 8, 100mM NaCl, protease inhibitor cocktail, 0,5% Triton X-100 and 0,1% SDS).
7) Amount of protein loaded:
For the first time we tried 25ug of protein. After, we performed a protein curve, using 20, 40 and 50ug of protein. We also tried to boil the sample to facilitate the binding of antibodies.
8) Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.):
Acrylamide gel 10% and 12%, SDS-page, electrophoresis for 2h / 2h30 at 80 / 100v.
9) Transfer and blocking conditions (Buffer/time period, Blocking agent etc.):
Transfer was made on ice for 2h at 350mA; 2h at 50mA; 2h30 at 85mA; 2h30 at 100mA; 3h at 100mA. Membranes were blocked with skim milk 5% for 15 minutes or albumin 5% for 30 minutes.
10) Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
DNM1L (ab93942) = 1:500.
Caspase 3 (ab32351) = 1:5000, 1:3000, 1:1500, 1:1000, 1:500.
APAF1 (ab2000) = 1:1000 and 1:500. All antibodies were incubated overnight, on ice, shaking. After, the membranes were washed with TBST 1X (6x for 5 minutes).
11) Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
The secondary antibody used was Goat pAb to Rb IgG (HRP) – ab6721. The concentration was 1:2000 for 1h on ice, shaking. After, the membranes were washed with TBST 1X (6x for 5 minutes).
12) Detection method (ECL, ECLPlus etc.):
First, we used Western Lightning ECL kit – Perkin Elmer. 500uL and 1000uL of each reagent and exposure for 1, 2, 3, 10 and 15 minutes.
We also used 500uL and 1000uL of ECL Western Blotting Substrate Kit (ab65628) for others tests and exposure for 15 seconds, 30 seconds, 1 minute, 1.5 minutes and 3 minutes.
13) Positive and negative controls used (please specify):
We used marker all the time and used an optimized antibody to check if our sample was ok.
14) How many times have you tried the Western?
For DNM1L we tried 5 times. For caspase 3 we tried 7 times. For APAF1 we tried 10 times.
15) Have you run a "No Primary" control?
No.
16) Do you obtain the same results every time? e.g. are the background bands always in the same place?
Yes. All the time we can’t see bands.
17) What steps have you altered to try and optimize the use of this antibody?
We changed all the steps of Western Blotting. We prepared new samples with two extraction methods, we changed the gel preparation, we changed the electrophoresis and transfer buffer, we changed blocking agent. We also checked if our samples in electrophoresis and transfer were ok, using Coomassie blue dying to visualize the proteins. We also tried different antibody dilutions as well as protein loading concentrations. |
ANSWER: |
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Thank you for contacting Abcam.
I am sorry about the problems that you have been having in western blot, with some of our products. Based on the information you have provided, I suspect that the problem is with the secondary antibody, since it would be really bad luck if you had all 3 primary antibodies fail to work, although for ab93942, we only predict that it will work in rat tissue, as we have not tested it rat yet.
I would be happy to send you a new secondary antibody to see if that resolves the issue. Also, under our Abpromise, both ab2000 and ab32351 is guaranteed to work in western blot on rat tissue, therefore if it does turn out that the primary antibody is at fault, then I would be happy to replace or refund the cost of these antibodies.
If you would like me to replace the secondary antibody, could you please provide me with the original order information for this product and also let me know if there is any particular secondary that you would like to receive.
I look forward to your reply and helping you to resolve this issue. |
