Lets say I have an antibody for HDAC5 which I will visualize, then incubate with GAPDH and visualize. After this step I would wash 2x in PBS and then could I add my next antibody of interest (TNF or Erk1/2) and incubate overnight? I do not have to strip because the MW are far enough apart...but I am wondering if I have to block before I add the next antibody of interest. I can use the GAPDH I know for all of them as a baseline.
If the proteins are different enough in size this is no problem. You don't have to re-block the membrane again (we usually used the antibodies in blocking buffer anyway). How do you visualize?
That is awesome news thank you...we use chemiluminesnce on a kodak imaging station
I would expect some signal from the first antibodies, but in general I don't see a problem. And you are not going to wash away the blocking, that needs a much harsher buffer. I used sometimes two different primaries at the same time, but this was on a Licor system with different secondaries coupled with different IR-dyes.
It's better that membrane should be cut two pieces, primary ab incubation is performed separately for each pieces
Hi, Jennifer, you can incubate 2 primary antibody sumultaneously. But if you use chemilumisnce detection, you shouild be sure about less or more equal signal for both antibodies. And, of course, as has been mentioned by Christian, have enough differences in size. Alternatively, you can use other system for detection of the second primary antibody: chemiluminiscnce for the 1st primary and NBT staininig for second primary. In this case you will never confuse antibodies! Good luck!
I agree with Meysam, to avoid nonspecific binding is best to cut the membrane
I believe blocking is necessary before adding second antibody of interest,otherwise the backgfround noise will increase if you are using chemiluminescence as the detection method. Cutting the membrane is also a good option, but i prefer mild stripping and then blocking and then adding of the second antibody of itnerest in order to avoid any background noise..
Pia Vida : sure, further , she can try each membrane with separated Primary Ab, and will see at one time after over-night incubation
When the primary antibodies are highly specific (what I assume), then it shouldn't make any difference if you cut the membrane in pieces or not. If they are not, its going to be a problem anyway.
I mean something else, according to far apart proteins of interest, she can cut the membrane for each primary Ab
@Meysam: I understood this, but why should you do it? It makes the handling more complicated.
@Jennifer: Yes. Preferrably you stain the membrane with a reversable stain like Poinceau Red (washes of with normal water afterwards) and then use a scalpell to cut the membrane. Make sure you add some corner or so, so you can puzzle the pieces back together. Or you load the marker in the middle and cut it in half.
I agree with the suggested answer of cutting the membrane into two parts if the bands of interest are far apart. This not only allows cleaner blot development, it is also a lot more efficient, since in the same amount of time, you now have two sets of bands to visualize in stead of one.
If you have to re-blot the membrane, then I would recommend an overnight washing step in PBS with very gentle shaking, followed by blocking and then the primary antibody. It has worked just fine for me in the past, since I try to avoid stripping the blot if I can.
- Badges
- Science method