hird1026, on 01 November 2012 - 06:31 PM, said:
1. if I use regular gels for probing, how does each lane matches each channel? The width is so different and it seems that some lanes might have more than one channel and some lanes might just locate in-between the channels. Is it possible to use miniblotter for a 15-well gel transferred PVDF membrane to perform the western blotting with different antibody each lane?
After the Ponceau S staining, you'll see red stain wherever you have protein, so you'll see the entire lane of proteins from your gel. If you mount the blot into the Miniblotter, you could use the red areas to see where the protein would be accessible to the antibodies you put in the wells. Then you can mark the wells of the Miniblotter that can bind protein (and not the lane divider areas), wash the Ponceau S stain out with water (I think that's what's used for wash; you'd probably want to check on that) and load your antibody in your normal blocking buffer into the wells that you marked.
You could probably set it so you have only one antibody probing each lane (the wells on the Miniblotter are certainly thin enough). You'll just have a band that's only as wide as the Miniblotter well when you develop your film (or on your scan).
I would suggest just marking where the wells on the blot when you have your gel sandwiched with the blot, but the gel often shrinks during Western transfer, so it may not be accurate enough for lining up with the Miniblotter wells.
The main issue here is being able to tell where the lanes/proteins are on your blot before mounting in the Miniblotter.
If you're using different protein samples for each lane, then I'd get some Ponceau S and use that to find the lanes, and line them up with the Miniblotter wells like I said. If you're using a single protein sample across the whole blot, but want to test many antibodies on it at once, then I'd just tear out the lane dividers and run a batch of protein across one big lane. It removes a lot of worries about lining up the blot (and you don't have to worry about residual Ponceau S stain messing with your detection method).
2. I am also looking for a method that could present total protein amount each lane on PVDF after transfer. Since my sample is ng-level, Ponceau S wouldn't be applicable due to the low sensitivity. Sypro Ruby might work but I couldn't find any detail protocol to guide me how it goes when I finish the staining and prepare for the blocking procedure of western. Does it require destain or any treatment of the PVDF membrane before I put it into blocking solution? Or could you suggest any possible method for my purpose?
Thank you or anyone willing to answer my questions!!
Oh, sorry, I originally missed the part about the amount of protein you're working with. If you think Sypro Ruby could work, then I'd make sure to specifically get the "Protein Blot Stain" version of that. I didn't see anything in the manual for the regular Sypro Ruby gel stain that indicates it's usable for blots. The Blot Stain manual indicates that you don't need to worry about washing the stain off, because ~90% of the stain will wash out in the blocking step (bottom-right of 2nd page): http://tools.invitro...als/mp11791.pdf
As I mentioned earlier, you could also try using a pen/pencil to mark the location of the gel lanes on the blot before transfer (or after transfer), and seeing if you can line up the Miniblotter wells that way. I would personally prefer the staining methods, as you can directly see where the proteins are, but if your protein quantities are that small, marking the lanes with a pen/pencil may be the only thing you can do.
If you do use Sypro Ruby, it may not be visible without a photo filter (I can't remember what it looked like the last time I used it), so you may need to visualize it and mark the lane locations then before blocking and mounting in the Miniblotter.
Hope that clarifies things a bit.