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We have tested the antibody for Dbx1 in both western blot and immunofluorescence (pictures attached).
In the wb there are a lot of unspecific bands. We have used mouse ES cells differentiated towards the motor neuron and at the recommended concentration and also higher. We have tested blocking with both milk and BSA. Also normal goat serum was used to preblock. As you can see with the Pcna, there is no degradation of the protein.
In the IF there is no staining at the right position and again unspecific staining where it is not supposed to be. The tissue is mouse embryo spinal cord E13. The dilution used was 1:1000. Antibodies for other proteins used at the same time worked without any problem.
Either there is a specific problem with this batch of antibody or there is a general problem with this antibody. In any case I don’t think the quality holds what promised.
Would appreciate your response
Asked on Mar 08 2012
Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.
I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been succesful. Reviewing the details, I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB, ICC-F and for mouse samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.
I can confirm that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed technical questionnaire below. To keep it less time consuming for now, I have just included a WB questionaire. I would appreciate if you could complete this. It will help you put the information we require together very easily.
Thank you for your time and cooperation. We look forward to receiving the completed questionaire.
Choose: Non-specific band Multiple bands No signal or weak signal High background
Purchase order number
or preferably Abcam order number:
Antibody storage conditions (temperature/reconstitution etc)
Description of the problem (high background, wrong band size, more bands, no band etc.)
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
Amount of protein loaded
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Detection method (ECL, ECLPlus etc.)
Positive and negative controls used (please specify)
Optimization attempts (problem solving)
How many times have you tried the Western?
Have you run a "No Primary" control?
Do you obtain the same results every time?
e.g. are the background bands always in the same place?
What steps have you altered?
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.
Answered on Mar 08 2012