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Anti-PTP mu antibody [2C10] (ab23879)

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1 question for ab23879

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Question 1

Thursday 17-August-2006

We received two antibodies which result in a high background on the Blot without any specific signal. Please find attached the first individual Western Blot questionaire form. Can you please give us some advice or suggestions?

ANSWER:

  

Thank you for taking the time to fill out our technical questionaire and send it in to us. I am very sorry you are experiencing difficulty obtaining satisfactory results from these antibodies (ab6721, ab23879) in your western blot procedure.

Thank you also for sending an image of your blots. This has been very useful, and I would like to make a couple of suggestions based on this that I hope will help. The blots are showing white (unstained) bands with a black (overstained) background. This is often indicative of using the antibodies at a concentration that is too high. I would therefore like to suggest the following:

1. Try using the primary antibody at 1:5000. Or at an even higher dilution if required.

2. Only one wash step has been mentioned in your procedure after primary antibody incubation. Try washing 3 or 4 times for 10 minutes each time. This should help wash away non-specifically bound antibody. It is good to include a detergent in the wash buffer, which you are doing.

3. Try blocking in 5% BSA which is often more effective at blocking non-specific binding. You can try incubating for 2 hours at room temperature.

4. The secondary antibody dilution looks like it should be fine. However, if you reduce the primary antibody concentration and still have black background, you may need to reduce the secondary antibody concentration aswell.

I hope these suggestions are helpful. Please don't hesitate to get back to me if you have any more questions or any more difficulties with the western blotting. Good luck!

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