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Cutting membrane after transfer - (Apr/01/2009 )

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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...

-rick112-

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

-mdfenko-

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.

-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

-yobou-

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

dom

-Dominic-

Hi

There is a simplest way to cat membrane (PVDF) directly on protein wells. I do this always after transfer. I do semi-dry transfer and after that transfer when you take the membrane and let them little dry you will be able to seen the all protein on membrane without any staining. But you mast be very careful and cat mambran quick. (Not to let them dry).

-asias-

yobou on Jun 15 2009, 07:33 AM said:

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


Nature Cell Biology ask for the whole blot. We recently published there and we had to provide almost every blot we showed in the manuscript

-laurequillo-

laurequillo on Sep 25 2009, 01:37 PM said:

yobou on Jun 15 2009, 07:33 AM said:

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


Nature Cell Biology ask for the whole blot. We recently published there and we had to provide almost every blot we showed in the manuscript


Once I have tried an instrument from BioRad Mini-PROTEAN II Multiscreen Apparatus (http://www.bio-rad.com/prd/en/US/adirect/biorad?ts=1&cmd=BRCatgProductDetail&vertical=LSR&catID=f9ec3085-b590-44c6-9b36-b36876cad466) which lets you to apply many different primary or secondary antibodies on a membrane with out cutting it it also need very low amount of antibodies.If it is possible you can also try this apparatus.

-banou-

Dr Teeth on Apr 2 2009, 07:10 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.


Why don't you just incubate the whole blot with all the antibodies simutaeously, as long as you know the apropiate concentrations of each antibody and you know which band corresponds to each antibody.

-Feelcontraire-

medchemgirl on May 20 2009, 12:30 PM said:

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.



To wash the ponceau from the membrane plain water or buffer, to remove it from the proteins 0,1M NaOH.

If you block with proteins, they usually take the ponceau with'em.

Coomassie is seen as an irreversible stain, thus it may impair antibody binding.

You may also let the membrane dry and the insert it in 20% ethanol, the membrane will turn translucent where proteins are (do it over a dark sourface or transilluminator).

-Feelcontraire-
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