Hello,
This time I want to ask two things about detection during western blot.
1) Casette, required for detection during western blot, is closed tightly
during detection so I think pressure is very important for detection.
In my latest detections in western blot, I found out that some part of casette is not located on bottom of table in dark room.
Some part was outside of table , which means that below this part of casette
there was no bottom. So I am concerned about difference of pressure.
Do you think this can affect detection results?
But I make sure that casette itself is closed tightly during detections
(P.S : To check whether casette is closed tightly, I check upper part of
casette by pressing very slightly some part of upper portion of casette during detection. Do you think this can affect detection results?)
2. I use different containers to pour developer and fixer respectively for detection during western blot.
I wonder whether some remnants in the containers can affect detection results
critically. (For example, waters used for washing but still remained in the containers) So far, it seems that there seems to be no problems
related to developer and fixer but it is just my subjective opinion.
I know these questions may be too broad or may seem a bit weird, but I am curious about questions above.
So I want to get opinions or answers.
Thank you!
Dear Lee
I agree with Christian
1. Generally uneven pressure in cassettes can lead to back ground problems; invariably darkened regions on your film or generally a darker haze on the film as a whole is the seal is not tight
2. ALWAYS use fresh water when washing fixer developer off your film; once again to reduce background and increase S:N
3. I will also add that you should be wary of using old developer: Developer with use and re use tends to photo oxidise and darken. Thus, limit use of the same aliquot of developer to a few hours during 1 day - this is acceptable; but do not leave on the bench exposed until the next day
4. Furthermore, keep fixative covered for RT storage and if you have a batch of fixative in its storage container that has darkened, preferably discard and open a fresh bottle of fixative
Use of old oxidised fixative will increase background but also reduce detection sensitivity
1.) If you make sure that the cassette in which you expose your film is closed correctly it should be fine. If the cassette was not placed fully on the table it does not influence your results. It is important that the film cannot move within the cassette.
2.) Make sure that the water you use for washing the film is fresh when you start your detection. Fixer and developer solution are usually concentrated rather highly so a little washing water that is transferred will have no effect on signal quality.
I hope this helps you.
Cheers, Christian
Dear Lee
I agree with Christian
1. Generally uneven pressure in cassettes can lead to back ground problems; invariably darkened regions on your film or generally a darker haze on the film as a whole is the seal is not tight
2. ALWAYS use fresh water when washing fixer developer off your film; once again to reduce background and increase S:N
3. I will also add that you should be wary of using old developer: Developer with use and re use tends to photo oxidise and darken. Thus, limit use of the same aliquot of developer to a few hours during 1 day - this is acceptable; but do not leave on the bench exposed until the next day
4. Furthermore, keep fixative covered for RT storage and if you have a batch of fixative in its storage container that has darkened, preferably discard and open a fresh bottle of fixative
Use of old oxidised fixative will increase background but also reduce detection sensitivity
Christian Q. Scheckhuber,
Thank you so much for your structured and informative reply,
I learned greatly thanks to your help.
I appreciate again.
Dear Laurence Stuart Hall,
Thank you so much for your great answer!
It seems that you also replied my previous questions, and that makes me grateful.
Hope for great achivements in your research.
Dear Lee
you are most welcome. Let me know if you have any further Western blotting questions
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