I have done this many times. When doing such short-time blocking and probing, I suggest room temperature, or even 37 degrees, depending upon the kinetics of your specific blot. What I suggest you do is do a test blot, if you can, of just a lane or two. Try blocking and probing at RT for an hour (each) on one and at 37 degrees for an hour (each) on the other. Then just go with whatever had the better empirical performance. I have done both methods, and it had to be determined empirically.
Yes, all the time.
Blocking 1h at room temperature.
However it also depends on your samples and quality of antibodies. You should try and then maybe you might need to increase the time abit if you get a lot of background compared to the overnight.
Yes, this can be done. I would suggest doing this with proteins that you know are fairly abundantly expressed. If you are probing for an rarer antigen, such as an unstable protein or a modified (ie phospho-, ubinquitinylated-) protein, you may require an overnight blot.
When I have done this, I have blocked for 1-1.5hrs at room temperature, blotted with the primary antibody for 2-3hrs at room temperature, wash 3x 15 minutes with your favorite wash buffer (I use 1x TBS with 0.2% Tween-20), blot the secondary antibody at room temperature for 1hr, wash 3x 15min again, develop.
If you reuse your primary antibodies (I reuse mine many many times as I keep them in 5% BSA in TBST and not milk), this treatment may decrease the lifetime of your antibody preparation, but sometimes time is of the essence.
Another thing that might be helpful is to block in 2X the strength of your normal blocking buffer. Not only does this cut down on time, but it also helps with background problems. Blocking at RT or 37C would definitely be a good idea if you’re only doing it for an hour.
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Yes, Iwhen I worked at a lab in NYU for the summer.
I have done this many times. When doing such short-time blocking and probing, I suggest room temperature, or even 37 degrees, depending upon the kinetics of your specific blot. What I suggest you do is do a test blot, if you can, of just a lane or two. Try blocking and probing at RT for an hour (each) on one and at 37 degrees for an hour (each) on the other. Then just go with whatever had the better empirical performance. I have done both methods, and it had to be determined empirically.
Yes, all the time.
Blocking 1h at room temperature.
However it also depends on your samples and quality of antibodies. You should try and then maybe you might need to increase the time abit if you get a lot of background compared to the overnight.
Yes, this can be done. I would suggest doing this with proteins that you know are fairly abundantly expressed. If you are probing for an rarer antigen, such as an unstable protein or a modified (ie phospho-, ubinquitinylated-) protein, you may require an overnight blot.
When I have done this, I have blocked for 1-1.5hrs at room temperature, blotted with the primary antibody for 2-3hrs at room temperature, wash 3x 15 minutes with your favorite wash buffer (I use 1x TBS with 0.2% Tween-20), blot the secondary antibody at room temperature for 1hr, wash 3x 15min again, develop.
If you reuse your primary antibodies (I reuse mine many many times as I keep them in 5% BSA in TBST and not milk), this treatment may decrease the lifetime of your antibody preparation, but sometimes time is of the essence.
Good luck.
As others have said, it can be done in an hour.
Another thing that might be helpful is to block in 2X the strength of your normal blocking buffer. Not only does this cut down on time, but it also helps with background problems. Blocking at RT or 37C would definitely be a good idea if you’re only doing it for an hour.