here are the 4 protein markers:
1) WT-1 , rabbit source, 52Kd
2) synaptopodin, goat, 100kd
3) POPX2, goat, 54kd
4) actin, rabbit, 42kd
I have heard about cutting the membrane and incubating it with primary antibodies separately, but let me just put it this way. As an undergrad im not allowed to do it this way. What i am required to do is incubate the entire whole membrane with each primary antibody at a time then incubate it with its appropriate secondary antibody, and then carefully load the substrate(chemiluminescence ECL) along the lanes corresponding to the protein marker ONLY and then detect. After detection of first marker, i will proceed with repeating all these steps for the second marker.
My plan would be to detect WT-1 first, then detect synaptopodin, then detect POPX2, then detect actin. Before detecting actin, i may need to treat membrane with stripping buffer because B actin primary antibody is derived from rabbit and its secondary antibody may react against WT-1 primary antibody(not to mention their sizes are pretty similar).
i have some questions which i hope people experienced doing western blot can answer.
1) is this antibody sequence of detection OK?
2) the detection of POPX2 might be interfered by synaptopodin (same source: goat) but i intend to load the ECL substrate onto the POPX2 protein size only. THE SIZE difference between these 2 should prevent ECL substrate from coming into contact with synaptopodin and giving me 2 detections during POPX2 detection?
3) I am concerned about this scenario: eventhough i am loading the substrate along the lanes corresponding to synaptopodin size only, and then detect this part of membrane, will the remnants of substrate from the first detection (WT-1) still be 'active' and give me another band?
4) this process may take me 4 days in total. ie: each marker incubation etc takes 1 day. will the membrane blot degrade and if so how do i ensure the membrane will be in good condition still?
5) alternatively i can just treat blot with stripping buffer after each step but my senior advise me that such treatments might affect the antigens, so its best to only use it when its needed.
Hello,
Here the main problem you will face: sometimes you will not get the protein band in the exact position or your protein might be denatured or multiple band will appear (for poly clonal antibody), therefore, it would be totally impossible to identify your desired protein band. Besides, you also don't know whether the primary antibodies you are going to use will interact in between them. Therefore, it is better to do westernbloting separately or cutting the membrane or re-blotting.
Best of luck
Thanks for the input. the interaction between primary antibodies is something that i had never thought of.
but if i cut, i might also run the risk of cuting away the desired band portion in case what you say may happen: that is protein migrate differently from usual due to denaturation.
any further advice is much appreciated!
- I agree with the first comment
- I would either blot separately or simply run a marker and leave a lane between groups of lysates for probing with each antibody; block whole membrane; cut with a fresh scalpel blade against each marker thereby ensuring your are not cutting into sample lanes; and then take each strip and incubate with primary antibody against these fresh replicate lysates
- DO NOT consider stripping and re probing the same lysate lanes with each of the 4 primary antibodies 4 times: stripping is invariably not complete and to aggressively strip to completely remove primary antibody you will probably begin to Denude proteins in your lysate lanes as well. This will reduce specific signal and increase background
- In addition if your first antibody yields a strong specific band compared to your next antibody by virtue of higher expression you are likely to end up with remnant signal from your first antibody in addition to signal from your second antibody. By the time you progressed to your 4th antibody with 3 rounds of stripping you could end up with a multi band output confusing interpretation
- Moreover a lot of antibodies in my experience yield low weight extra specific bands due to in vivo breakdown of protein if interest; other non specific bands due to cross reactivity and extrinsic in vitro breakdown products of your specific band due to breakdown of your specific protein during lysate preparation. In addition particular tissues can yield extra specific bands representing other protein isoforms created by tissue specific alternative splicing : all such confounding factors are an attribute of your tissue and an artifact of particular lysate preps. With multiple antibodies this could simply add to the confusion
- Thus perform separate western blots fir each primary antibody
Yes. you shouldn't cut the same membrane for each of the four different protein. It would be best if you blot separately. However, agreeing with Dr. Hall I would suggest you to reblot your membrane highest ones that means you will get two protein bands from a single membrane separately. Hope, this might help you.
Best regards
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