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Product code: 76424
Lot number: GR81000-1
1)Antibody storage conditions (temperature/reconstitution etc): the antibody was stored at -20oC
2)Description of the problem (high background, wrong band size, more bands, no band etc.): No band detection
3)Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.): whole cell lysate ZL55 e ZL34 cell lines
4)Sample preparation (Buffer/Protease inhibitors/Heating sample etc.): RIPA buffer plus Protease and Phosphatase inhibitors
5)Amount of protein loaded: 25 and 50 ug
6)Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.): Denaturing SDS PAGE (12%)
7)Transfer and blocking conditions (Buffer/time period, Blocking agent etc.): Semi-dry transfer Blocking: 5% milk prepared in TBS+Tween (0.01%)
8)Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
Primary antibody: anti-survivin primary antibody (Abcam cat n° ab76424). diluition 1/2000 and 1/1000 in blocking buffer Incubation time: over night at 4°C Wash step: TBS+Tween for 10 minutes (three times)
9)Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): Secondary antibody: anti rabbit pierce diluition 1/2000 in blocking buffer Incubation time: 1 hour at room temperature Wash step: TBS+Tween for 10 minutes (three times)
10)Detection method (ECL, ECLPlus etc.): Supersignal West femto chemiluminescent substrate (Thermo Scientific).
11)Positive and negative controls used (please specify): whole lysate from HeLa cells
12)How many times have you tried the Western?: three times
13)Have you run a No Primary control?: no
The customer has no images because there was no signal at all.
Asked on Oct 16 2012
Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear the customer has had difficulty obtaining satisfactory results from this antibody.
The details you have kindly provided will provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee for WB and human samples.
I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggest you may have regrettably received a bad vial.
I can recommend you may like to consider trying the following options:
1. Could you confirm if the transfer to the membrane and quality of the sample has been assessed using a loading control?
2. Is the current vial of secondary antibody working well with other rabbit antibodies?
3. I can suggest to try using BSA rather than milk to block. Changing blocking agents can sometimes help to improve the results.
Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.
Answered on Oct 16 2012