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Dear Ms Hayes,
Please see my answers within the questionnaire below.
Antibody code: ab61488
Choose: Non-specific band Multiple bands No signal or weak signal High background
Lot number GR35985-1
Antibody storage conditions (temperature/reconstitution etc) antibody aliquoted and stored at -20 according to manufacturer's recommendation upon arrival.
Description of the problem (high background, wrong band size, more bands, no band etc.) Non-specific band Multiple bands No signal or weak signal High background
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Total protein extract from mouse embryonic stem cells differentiated towards motor neuron (embryonic bodies). Testing for endogenous protein. We have confirmed that the gene is expressed in these cells by RNA-seq and RT-qPCR, repeated several times.
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Normal cell lysis buffer with medium stringency (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100) complemented with protease and phosphatase inhibitors from Roche. Samples mixed with sample buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue), heated at 70 for 10 min.
Amount of protein loaded 20 ug of sample loaded
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) NuPAGE Novex 4-12% Bis-Tris Gels under denaturing conditions, 180V for 1.5hrs
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Tris-glycine buffer for 2-3 hrs at 65V, blocked with 5% BSA first time and 5% milk second time.
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Dbx1 from Abcam rabbit, diluted 1:1000 first time and 1:500 second time in 5% BSA with 1% and 2% normal goat serum o/n at 4. Washed 3 x 10 min with PBS-Tween 0.5%
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Calbiochem Goat anti rabbit diluted 1:5000 in 5% BSA incubated 1 hr at RT followed by 3x5 min wash in PBS-T and 1x5 min in PBS
Detection method (ECL, ECLPlus etc.) ECL
Positive and negative controls used (please specify) As mentioned above, samples have been tested by RNA-seq
Optimization attempts (problem solving)
How many times have you tried the Western? 2 times
Have you run a No Primary control?
No, but we have not had with this secondary which we have been using for the past 3 years
Do you obtain the same results every time?
e.g. are the background bands always in the same place? Yes
What steps have you altered? Blocking, ab concentration
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.
Asked on Mar 09 2012
Thank you for taking the time to complete our questionnaire. The details you have kindly provided will provide us with vital information for our monitoring of product quality.
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 are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.
I apologise for the inconvenience and 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 Mar 09 2012