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I filled out the questions the best I could, I hope you can find some conditions I could improve.
Thank you very much for your time,
Antibody code: ab19867
Choose: Non-specific band Multiple bands (and the correct one is missing, so I guess that also makes it weak signal?)
Antibody storage conditions (temperature/reconstitution etc)
AB reconstituted in 100% glycerol, stored at -20
Description of the problem (high background, wrong band size, more bands, no band etc.)
Expected band (ca 43kDa according to data sheet) not seen; persistant band at ca 60kDa
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
cell extract from human CRC cell lines (LS180 and HCT116; HCT116 is known to have no MEIS1 RNA, and thus not expected to have MEIS1 protein, LS180 does express MEIS1.)
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
10%SDS with complete inhibitor and PHOSstop (Roche), samples heated 5min at 100 degrees after isolation. Re-heated just before loading (and adding of leamli buffer)
Amount of protein loaded
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
10% bisacrylamide with 0,1% SDS
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
blot buffer : Bio-rad 161-0771. transfer for 1 hr at 100V; largest proteins on marker (250kDa did transfer, so no reason to expect MEIS1 not to be transferred)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
MEIS1 Rb polyclonal, diluted in TBS-T (0,05%Tween) +5% BSA/dried milk
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Gt anti Rb ::HRPO 1:10.000 in TBST, 1hr at rt, washed 4x for 15 min in TBST lightly shaking
Detection method (ECL, ECLPlus etc.)
Positive and negative controls used (please specify)
non-primary antibody control (in the picture columns A1 and B1)
No positive control taken along, but same prot. Lysates were used for other blots, and showed no signs of degradation.
Optimization attempts (problem solving)
How many times have you tried the Western?
BSA vs dried milk (both at 5%) (=first test, detected with ECL and incubated with primary AB for 1 hr at RT)
Showed less signal then the 3rd test, but patern was similar
Nitrocellulose membrane with odyssey labeling as detection method (=second test, primary AB for 1hr at RT)
Only 60kDa band was visible for the 1:2000 and 1:4000, but very faint, and for 1:1000 many bands appeared
Primary AB O/N at RT (=3rd test, of which the picture is included; detection with ECL, membrane = PVDF
See result in included picture, longer AB incubation results in more aspecific bands; and no band at the expected size.
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?
60kDa band always appears
What steps have you altered?
membrane (PVDF vs nitrocellulose, detection method; odyssey (using ALEXA-labeled secondary AB) vs ECL, primary AB incubation for 1hr or O/N
enclosed picture is with ECL with 30 seconds exposure; PVDF membrane, o/n incubation with primary AB at RT,
Asked on Aug 29 2012
Thank you for taking the time to complete our questionnaire and contact us. 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 like to offer some suggestions to help optimize the results I would also appreciate if you can confirm some further details:
1. I am sorry I am not able to trace this order number on our system, and there are no orders from the Netherlands in March or April this year. Please confirm if the product code is correct (ab19867 Anti-MEIS1 antibody - ChIP Grade) and please provide the name and address of the institution where the order was placed from. If you ordered from a purchasing agent somewhere else, then they should have the Abcam order reference number.
2. We recommend to store antibodies as directed on the datasheet so that they will remain stable and that we can continue to provide our guarantee. If addition of glycerol is required, this will be mentioned on the datasheet. For future reference, this antibody should be aliquotted and stored as it is, as directed on the datasheet. Extra glycerol is not required in this case. Storing an antibody at lower concentrations can affect its stability.
3. I can recommend to lyse the cells in RIPA lysis buffer, which should provide a more suitable protein preparation. Once the lysate is prepared, you can then add sample buffer including reducing and denaturing agents, and heat.
4. What dilutions of antibody have been tried? To reduce the background, I c an recommend to try reducing the concentration of antibody. Incubate overnight at 4oC, which can provide more specific and efficient staining.
5. I can recommend to add a wash step after the primary antibody incubation which will help to wash away any excess antibody.
6. Could you confirm the current vial of secondary antibody is working well with other primary antibodies? I suggest it would be beneficial to include a no primary control to assess if the secondary antibody is binding non specifically. It may be that the concentration of secondary antibody needs to be reduced in order to help optimize the results.
7. We recommend not to mix blocking agents in an experiment, it can sometimesaffect the results. Try 5% BSA only.
8. Has the quality of the sample and transfer to the membrane been assessed using a loading control?
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 Aug 29 2012