
Cutting membrane after transfer
#1
Posted 01 April 2009 - 10:00 PM
Is it possible to cut a membrane after transfer, to incubate the different sections with different primary antibodies?
What is the best way of doing this without damaging the membrane?
#2
Posted 02 April 2009 - 12:03 AM
I usually do this rather than stripping the membrane. Just stain with ponceau red to check how the lanes look and cut accordingly with a regular scissor.
In case I use an antibody the very first time I do not cut the membrane but try to see how specific it works by offering the complete membrane and not only the piece of expected molecular weight.
#3
Posted 02 April 2009 - 06:10 AM
Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley
#4
Posted 02 April 2009 - 03:53 PM
Our lap routinely slices blots horizontally using prestained markers and stain each separate blot with different primary antibodies. It works great and saves you from needing to strip/restain blots which is time consuming and sometimes too ineffective or too harsh. As long as you know your antibodies are specific, I don't see anything wrong with this. People who stain the whole blot only show a strip anyway for a publication figure.
Many journals now ask for a full picture of the blot to be included in supplementary information, to make sure that the blot is as the authors said it is.
#5
Posted 03 April 2009 - 06:56 AM
Our lap routinely slices blots horizontally using prestained markers and stain each separate blot with different primary antibodies. It works great and saves you from needing to strip/restain blots which is time consuming and sometimes too ineffective or too harsh. As long as you know your antibodies are specific, I don't see anything wrong with this. People who stain the whole blot only show a strip anyway for a publication figure.
Many journals now ask for a full picture of the blot to be included in supplementary information, to make sure that the blot is as the authors said it is.
If they ask for it, then run another experiment to obtain a whole blot or use one of your initial experiments as you still save money in the interim. As long as you are not using this procedure to hide anything, I don't see the problem. As I said, BEFORE using this technique, do a few experiments where you stain whole blots too ensure that your antibodies are specific. Besides, I've published numerous papers and JBC, Mol Pharm, Toxicol Sci, Cell, etc. have never asked for whole blot pictures.
Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley
#6
Posted 07 April 2009 - 05:28 PM
I recently had Oncogene ask for a full blot, mostly I suspect because the reviewer is a competitor in our field. I also do the strip thing, just not for publication blots.If they ask for it, then run another experiment to obtain a whole blot or use one of your initial experiments as you still save money in the interim. As long as you are not using this procedure to hide anything, I don't see the problem. As I said, BEFORE using this technique, do a few experiments where you stain whole blots too ensure that your antibodies are specific. Besides, I've published numerous papers and JBC, Mol Pharm, Toxicol Sci, Cell, etc. have never asked for whole blot pictures.Many journals now ask for a full picture of the blot to be included in supplementary information, to make sure that the blot is as the authors said it is.
#7
Posted 20 May 2009 - 11:30 AM
Yes, sure.
I usually do this rather than stripping the membrane. Just stain with ponceau red to check how the lanes look and cut accordingly with a regular scissor.
In case I use an antibody the very first time I do not cut the membrane but try to see how specific it works by offering the complete membrane and not only the piece of expected molecular weight.
#8
Posted 20 May 2009 - 02:46 PM
#9
Posted 21 May 2009 - 07:15 AM
talent does what it can
genius does what it must
i used to do what i got paid to do
#10
Posted 21 May 2009 - 01:07 PM
#11
Posted 24 May 2009 - 08:10 AM
also about complete transfer..hmm..tricky thing depends lot on protein..some do get transfer well enough that u will not detect much in gel (may find fine band if u silver stain the transfer gel..)..while others are little tricky...
#12
Posted 26 May 2009 - 12:34 PM
talent does what it can
genius does what it must
i used to do what i got paid to do
#13
Posted 14 June 2009 - 09:12 PM
Edited by yobou, 14 June 2009 - 09:13 PM.
#14
Posted 14 June 2009 - 09:33 PM
could you please name some of these journals?Our lap routinely slices blots horizontally using prestained markers and stain each separate blot with different primary antibodies. It works great and saves you from needing to strip/restain blots which is time consuming and sometimes too ineffective or too harsh. As long as you know your antibodies are specific, I don't see anything wrong with this. People who stain the whole blot only show a strip anyway for a publication figure.
Many journals now ask for a full picture of the blot to be included in supplementary information, to make sure that the blot is as the authors said it is.
my lab publishs in journals like cancer research, oncogene, mol cancer ther, mol cancer res, carcinogenesis, cancer science and we were never asked to show the whole image of the blot or gel. I did not expect that peer review means the authors are assumed to fabricate the results!!! but rather is concerned with the validity of conclusions with respect to experimental design
Edited by yobou, 14 June 2009 - 09:36 PM.
#15
Posted 15 June 2009 - 05:31 AM
dom