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Product code: 124905
Lot number: GR78448-2
Inquiry: Antibody code: ab124905
Batch number: GR78448-2
Antibody storage conditions (temperature/reconstitution etc): -20oC
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.):
total cellular proteins purified from Emiliania huxleyi, proteins isolated from human fibroblasts cells that strongly express the protein were used as positive control (other tests on this sample showed that it indeed possesses the protein). No bands were detected in the positive control.
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.): Tris pH=8, EDTA, SDS, Protease inhibitor.
Amount of protein loaded: 30ug
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.): Reducing 4-16% precast Criterion TGX gel (BioRad)
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.): As described by BioRad. Identical gels were used for WB with a different antibody (ab4753) that worked well
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): ab124905 1:2000
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): Goat anti Rabbit HRP (Jackson 111-035-003)
Detection method (ECL, ECLPlus etc.): ECL Plus Positive and negative controls used (please specify):
Positive control – proteins isolated from human fibroblasts cells that strongly express the protein were used as positive control (other tests on this sample showed that it indeed possesses the protein).
OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)
How many times have you tried the Western? 3
Have you run a "No Primary" control? yes
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? Increasing concentration of primary antibody, Increasing incubation time with primary antibody, increasing protein concentration on the gel.
Asked on Jul 26 2012
Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have 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 ab124905 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 like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some further details:
1. I can suggest to try RIPA rather than Tris buffer to lyse the samples. This may provide a more suitable protein preparation.
2. Try the antibody at a higher concentration to increase the signal, for example 1:1000 or even 1:500. Incubating overnight at4oC can often provide more specific and efficient staining.
3. Has the transfer to the membrane and quality of the sample been assessed using a loading control?
4. Is the current vial of secondary antibody working well with other primary antibodies?
I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.
Answered on Jul 26 2012