Novel(?) Western Blotting question

[fitbrit]

EDIT: It worked!


Hi all- what a great resource this is!

Short form of my question:
Can multiple samples be loaded on a single well for Western blotting with a time delay between each? The TOTAL amount of protein loaded can be kept the same as usual by reducing the volume we load.


Same question in-depth with background:
I'm interested in quantifying the appearance of a ~40 kd protein in response to a certain treatment of a cell line. The vast majority of Western blots my group does is on this protein. Our antibody yields a very clean result and is highly sensitive should we choose to do things a certain way.
Recently, I've been thinking that loading multiple (up to three) samples on the same well at 0', 40' and 80' (for a total running time of 120') might be a good way to visualise the results of our experiments, and of course to save on consumables (we use precast gels and an iBlot transfer system).
We could load multiple experiments across the same gel, or multiple conditions on the same well etc.
Since the primary antibody yields very clean results, it should work right? I'm aware that we have to be careful about overloading wells, but we can easily cut downt he amount of protein we load to 1/3 of what we use currently. Of course, we'd also reserve three wells with a positive control to be loaded singly at the same time as the second and third loading of the experimental wells. We could even go so far as to load negative controls in the three control wells too to ensure every well gets the same total amount of protein at every stage of the running. I hope you're all still with me!

Can anyone suggest any flaws in the idea, or any reasons why we won't be able to get useful data out of it. I understand that in general this is a bad idea, but for my specific purposes it might be feasible.

Thanks in advance. I'll be happy to add info as needed.

[danfive]

Hi very innovative idea! If you can pull it off, go for it. I assume you are not using gradient gels.

[fitbrit]

Thanks, Danfive... we're not using gradient gels right now, but I thought that it might work better if we did... to get better spacing of the three sets of bands. P.S. I should add that I'm the group leader, so it's no problem going ahead to try it at the moment. I'll keep you all posted on how it goes.

[kmunson779]

If your gels are the standard Laemelli recipe (different pH stacking and running gels) then it would probably be a bad idea; the stacking gel would only work for the first sample, then the later samples wouldn't get stacked, leading to wide, blurry bands.

ETA: Also, would your loading control be well enough separated from your POI bands to cut the membrane properly? Is your loading control antibody as clean as your POI antibody?

[danfive]

As an afterthought, your situation may be ideal for dot blotting or slot blotting. That is if you don't need size separation in addition to the antibody binding.

[fitbrit]

Originally Posted by kmunson779

If your gels are the standard Laemelli recipe (different pH stacking and running gels) then it would probably be a bad idea; the stacking gel would only work for the first sample, then the later samples wouldn't get stacked, leading to wide, blurry bands.

ETA: Also, would your loading control be well enough separated from your POI bands to cut the membrane properly? Is your loading control antibody as clean as your POI antibody?

The gels are not standard Laemelli ones; they're pre-cast Invitrogen Bis-Tris gels with no stacking portion. The loading control is actually the non-phosphorylated POI, (the POI is the phosphorylated form) and will be determined by stripping and redetecting. So no problem there hopefully, and no need to cut the membranes.

[fitbrit]

Originally Posted by danfive

As an afterthought, your situation may be ideal for dot blotting or slot blotting. That is if you don't need size separation in addition to the antibody binding.

Yes, that could be an even nicer solution. I'll look into it also. Thanks for pushing me out of my gel-centric view of the world!
One of my team actualy has tried both methods today and we'll now the results tomorrow. I'll let anyone reading this thread know.

[fitbrit]

Follow-up:

My idea worked perfectly. This is really very good news for us. The dot blots were thrown together at the last second and didn't work, but we were really just guessing at everything there. Tomorrow we'll try both techniques again.
Thanks everyone for your input.