27 replies to this topic

[asias]

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).

[laurequillo]

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

Edited by laurequillo, 25 September 2009 - 03:37 AM.

[banou]

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.c...36-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.

[Feelcontraire]

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]

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.??

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).

[goldfinger]

I do this all the time. Especially when you are blotting with different phophorylation antibodies at the same time this is the only way to do the work fast and efficient. I cut up to 10 pieces of my PVDF using a razor blade.

[scifistudent]

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

dom

we also use dual marker from Biorad. And i saves our time from strip and ponceau etc.

we are going cool with it.

good luck

[acquire]

phophorylated proteins we should check first then non phosphorylated or vice versa?for eg p-AKT first or Akt (both r having same mol wt 60kd.) like these cases what u suggest.

[science noob]

phophorylated proteins we should check first then non phosphorylated or vice versa?for eg p-AKT first or Akt (both r having same mol wt 60kd.) like these cases what u suggest.

I heard stripping would remove some proteins as well, so would it be advisable to reprobe similar sized proteins (e.g. both at 60kD)?

[PhilB2442]

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

I've had similar experience with NCB. They want to see the whole blot for most of the westerns included. I've heard of a reviewer asking to see each blot of replicates from a densitometry graph as well.

[PhilB2442]

phophorylated proteins we should check first then non phosphorylated or vice versa?for eg p-AKT first or Akt (both r having same mol wt 60kd.) like these cases what u suggest.

I usually check phosphorylated first because the antibodies tend to have slightly lower affinity for their targets than the total. For something like AKT, though, it really doesn't matter. I use pAKT antibodies from cell signaling (total, S473, and T308) and they all work extremely well.

[Genecks]

I'm sorry. I'm having trouble understanding what all of you are talking about when you're saying that you're cutting the membrane up.
Some pictures would help or descriptive language.

I can think of it like this:

Say I have 6 lanes:
| Seeblue plus 2| sample | sample | sample | sample | sample |

It's the same sample. Seeblue plus 2. Let's say I do a 12% gel, and I run it until the bands space out pretty well for me to probe for the proteins.

Let's say that I'm testing for actin, amyloid precursor protein, presenilin-1, beta-catenin, and gamma-secretase (so, five different proteins).

Afterward, I take apart the gel, blot it onto nitrocellulose paper, and check it with ponceau S to see if I have bands.
I use PBST to wash off the Ponceau S, and then...

Ok, so let me see if I understand what all of you are talking about...

Then, what I do, is I cut paralell to the direction the bands have sorted out by length in each of their respective lanes, without cutting through the protein lanes.
And then I have the opportunity to place each strip with its respective protein bands into its own plastic bag with antibody.
Afterward, I piece everything back together in a film casette, bring it to the film room, and attempt to develop.

Now, that sounds like a great idea, but I would definitely would have to wait different time intervals for the bands to appear. And wouldn't there be some over-exposure? For instance, the actin band would get super dark over time, right? Maybe messing with the film?

Edited by Genecks, 05 May 2012 - 07:01 PM.

[mdfenko]

you can expose them all separately then piece the resulting images together (or present them separately).