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Product code: 62336
Lot number: GR69244-2
Inquiry: Dear Tech Support Team, Please read below the details of customer's complaint. Please advise. Thanks in advance. )
Abcam product code ab62336
2) Abcam order reference number or product batch number lot # GR-69244-2
3) Description of the problem - Did not recognize protein in 3 cell types including recommended positive control (NSC34, Cos7, Hela)
4) Sample preparation: Type of sample: whole cell lysates Lysis buffer : 1% triton / 1% NP40. Protease inhibitors: complete (prot. inhibitor cocktail by Roche) Phosphatase inhibitors : Phos-stop Reducing agent β-ME
Boiling for ≥5 min? yes/no
Protein loaded ug/lane or cells/lane – 30-50ug.
Positive control Hela cell lysates
5) Percentage of gel 12%, 10%
Type of membrane nitrocellulose Protein transfer verified Yes – marker, ponceu
Blocking agent and concentration 5% milk in TBST Blocking time 1hr Blocking temperature room temp’
6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution 1:200, 1:1000, 1:500 Diluent buffer 5% milk Incubation time Over night Incubation temperature: 4°C
7) Secondary antibody: Species: Donkey Reacts against: rabbit Concentration or dilution 1:1000 Diluent buffer milk / TBST Incubation time 1hr Incubation temperature: room temp. Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies: Buffer TBST Number of washes 3 X 5’
10) How many times have you run this staining? 3 Do you obtain the same results every time? yes What steps have you altered to try and optimize the use of this antibody? cell type, dilution, ug protein. Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem. Nothing on the film. other membranes in the same exposure did give results. Half of the same membrane incubated with a different Ab did give result.
Have a nice day.
Asked on Jul 26 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 enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.
I would like to reassure you that ab62336 is tested and covered by our 6 month guarantee for use in WB and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.
Reviewing this case, I would if you can confirm some further details in order to help me investigate this further:
1. Please confirm further details of the results. Was the membrane completely clear, or was there background staining, or non specific bands at incorrect size? I would appreciate if you are able to provide an image, including molecular weight markers, which will help us to assess the results.
2. The antibody detects the phosphorylated (S24) stathmin 1. As far as I am aware, the phosphorylation can require inducing. For example, the HeLa cell lysate used in the WB shown on the datasheet has been treated with FBS+Calyculin A. Could you confirm if the cells used in these experiments have been treated? I can suggest considering a further literature search for more information.
3. Has the transfer to the membrane and quality of the sample been assessed using a loading control.
I hope this information is helpful. Please do not hesitate to contact me again with the further requested details and I hope we can resolve this case as soon as possible.
Answered on Jul 26 2012