28 replies to this topic

[Pipette Dude]

Hi Dudes,

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?

[Bomber]

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.

[Dr Teeth]

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.

[bob1]

Dr Teeth, on Apr 2 2009, 06:10 AM, said:

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.

[Dr Teeth]

bob1, on Apr 2 2009, 07:53 PM, said:

Dr Teeth, on Apr 2 2009, 06:10 AM, said:

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.

[bob1]

Dr Teeth, on Apr 3 2009, 07:56 AM, said:

bob1 said:

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.

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.

[medchemgirl]

Im wondering, where do you do ponceau red on, on the membrane or on the gel?. If the proteins where transfered completely u wont see anything on the gel. If you stain the membrane, how do u clean it then for blocking, etc.??

Bomber, on Apr 2 2009, 02:03 AM, said:

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.

[dtimm]

Ponceau your membrane.  It comes off pretty well in washing buffer.  I commassie the gel if I need to check transfer efficiency.

[mdfenko]

ponceau will wash off of the membrane with water if you don't want to use buffer.

[medchemgirl]

Cool, I will try that then. Will it work the same with commassie blue?

[rick112]

yes it will work with commassie also..u can cut the specific region destain it and use for any other purpose...
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...

[mdfenko]

coomassie won't wash off with water (at least, not as easily nor as quickly as ponceau s).

[yobou]

I always cut the PVDF membrane and probe each piece with a different primary Ab. To do this I load the marker on the first and last lanes of the gel, then after blotting wrap the membrane in Saran wrap then draw lines along the cut site gently with a marker pen then cut with sissor.

Edited by yobou, 14 June 2009 - 09:13 PM.

[yobou]

bob1, on Apr 3 2009, 08:53 AM, said:

Dr Teeth, on Apr 2 2009, 06:10 AM, said:

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.

could you please name some of these journals?
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.

[Dominic]

try using a coloured ladder and simply cutting thru the center of that - saves on staining

dom