Concurrent detection of two proteins?

[ChocoCowie]

In western, if I need to detect two proteins, can I expose the nitrocellulose membrane to two different primary antibodies. So the proceedure would be
- transfer
- block
- first primary
- second primary
- first secondary
- second secondary
Reason I don't seperate the membrane and expose the different strips to different Ab's at the same time is because the two are very close to together

Thanks!

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[xanthines]

I would be very careful about doing this. Make sure each antibody results in one band, ie no non-specific signals. Also, makre sure the signals aren't too strong for each protein, otherwise you will get one big whopping band which would be useless.

Why don't you just run duplicate lanes (or gels if you wish), and cut the membrane vertically?

-X

In western, if I need to detect two proteins, can I expose the nitrocellulose membrane to two different primary antibodies. So the proceedure would be
- transfer
- block
- first primary
- second primary
- first secondary
- second secondary
Reason I don't seperate the membrane and expose the different strips to different Ab's at the same time is because the two are very close to together

Thanks!

 

[Belfagor]

Why don't you just run duplicate lanes (or gels if you wish), and cut the membrane vertically?

-X

That's solid. Easier than worrying about getting a big blob. Spacing can be a pain.

 

[vcatz]

You could always do the western with the first primary then strip the blot and re-probe with the second primary. Pierce sells a gentle stripping buffer that is good at removing antibodies but not protein from the membrane.

 

[tr]

Amersham and Invitrogen sell fluorescent probes for Western. I haven't used them myself, but I imagine it might be possible to double-probe with different colors.

 

[grendel]

Amersham and Invitrogen sell fluorescent probes for Western. I haven't used them myself, but I imagine it might be possible to double-probe with different colors.

quantum dots! pretty neat stuff.

 

[linuxizer]

Like xanthines said, you can do it on the same membrane as long as the antibodies both produce clean results and the molecular weights are different enough. Otherwise, if they are dirty but different MW, run a second marker (diluting it helps you tell which side is which) as the last lane and cut horizontally. Otherwise, run a duplicate lane and cut vertically (it helps to run some marker in between to see where to cut) or strip and reblot.
Good luck,
Ari