Anti-ATM antibody [2C1 (1A1)] - BSA and Azide free (ab78)

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 like to offer some suggestions to help optimize the results from ab78. I would also appreciate if you can confirm some further details:

1. I would appreciate if you are able to provide an image, including molecular weight markers, which would help me to assess the results.

2. Please confirm which mouse cell lines have been tested?

3. I can suggest the cells need to be fully lysed in lysis buffer such as RIPA. SDS loading buffer alone will not necessarily provide good lysis and protein preparation.

4. I can also suggest toinclude protease inhibitors in the lysis buffer as smearing down the lane on the blot indicates the proteins in the sample may have degraded. For the same reason, I can suggest to try fresh samples and reagents if possible.

5. I recommend to check the amount of protein being loaded, to ensure the gel is not overloaded. We recommend to do a protein assay on each sample and to load 20 - 30 ug of total protein per lane of the gel.

6. Smearing may also indicate the gel has been run too quickly, particularly with a larger protein. Try reducing the voltage and increasing the gel running time which should help provide a better resolution.

7. Reducing the concentration of antibody will help to reduce any background. Try 1:2000 as recommended on the datasheet.

8. I can recommend these further tips for transfer of larger proteins, from our protocols pages:

Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation.
Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily.
Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich.

9. Could you confirm ifthe secondary isworking well with other primary antibodies? What are the results of the no primary 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 information and I will be pleased to provide the alternative free of charge replacment

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 would like to reassure you that this antibody is tested and covered by our 6 month guarantee for western blotting, and for Mouse, Rat, Human and Monkey 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. Alteratively, as requested we can arrange a free of charge replacment of an alternative antibody.

Although the other antibodies have worked well using this procedure, individual antibodies will often require optimization. Before deciding how to proceed in this case, I would like to investigate this further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. 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.



Order Details
Antibody code:
ab78 -Mouse monoclonal [2C1 (1A1)] to ATM


Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
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?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes: