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please see below. I don’t think the antibody ab412 works correctly.
Antibody code: ab412
Choose: No signal
Lot number GR104284-1
Purchase order number
or preferably Abcam order number:
Antibody storage conditions (temperature/reconstitution etc) -20°C
Description of the problem no signal in whole cell extracts.
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
N2a cells (murine cell line), whole cell extract
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
Lysis in 50 mM Tris-HCl 8,0 + 140 mM NaCl + 0,25% NP40 + protease inhibitor mix
Sample preparation: 10 µl sample + 4,7 µl Reducing loading buffer (Fermentas), 5 min 95°C
Amount of protein loaded
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
10% denaturing, non-reducing gel
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Tank Blotting 1 h 150 mA (Biorad)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
1:1000 ab412 in PBS/milk
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
1:10.000 anti-mouse-POD (A4416, Sigma)
Detection method (ECL, ECLPlus etc.)
ECL-Plus (Perkin Elmer)
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?
Yes No Yes
Do you obtain the same results every time?
Yes No Yes
e.g. are the background bands always in the same place?
What steps have you altered?
different amount of protein used for IP / input
More antibody ab412 used for IP
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to assess the results.
see attached file, lane 1, input A; Lane 2, input B; lane 3, eluate A; lane 4, eluate B
Asked on Dec 05 2012
Thank you for taking the time to complete our questionnaire. 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.
Reviewing this case, I would appreciate if you can confirm some further details:
1. Could you confirm if reducing and denaturing conditions have been tried? We recommend to use antibodies in reducing and denaturing conditions in WB unless it recommends to use non denaturing and reducing conditions on the datasheet. This will ensure that the proteins are in the correct conformation to run at the correct molecular weight and be detected by the antibody.
2. Could you confirm what size are the 3 bands in the blot in the eluted samples? And what size bands were you expecting?
3. How was the sample eluted from the IP column? Which buffer was used?
4. Which lanes are the whole cell extract in, is this lane 1 and 2 (input A and B?).
5, Has the transfer to the membrane and quality of the sample been assessed with a loading control in the whole cell extracts? Transfer to the membrane may be different from the eluted samples if they are in different buffers etc).
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 Dec 05 2012