3 replies to this topic

[hird1026]

I read that Miniblotter (28 channels, Immunetics) could be used for multiple western blot on the same PVDF membrane after transfer.
My question is how do I align each well of 15-well PVDF membrane (transferred from a mini gel) with the 28 channels and perform the western without cut the membrane into strips?

Thanks !!

Edited by hird1026, 01 November 2012 - 03:03 AM.

[John Forsberg]

I've used the Miniblotter for probing sets of hybridoma supernatants for screening during monoclonal antibody production.

Generally speaking, it works best if you have one molecular weight standard lane to one side, then one big well running across the rest of the gel that you load with your protein sample/lysate.  That way you can cover as many of the Miniblotter wells as possible without worrying about whether a particular well is binding in the gap between lanes.

If you pour your own gels, you can simply take a piece of Scotch tape and tape around all of the wells but one, so when your gel solidifies, it leaves one long well instead of several.  You just load your sample in one huge volume instead of several smaller ones.  I usually ended up using about 50 µg per normal lane, or about 500 µg across a whole long lane.

Then when you line up your blot in the Miniblotter, you can line up your antibody wells with your large lane.

As we use commercial pre-cast gels most of the time, I just got the single-well pre-cast gels from Invitrogen, then cut a lane divider off another regular gel, and wedged it down between the 1-well gel plates to make a quick and dirty version.  You could probably also get away with cutting the lane dividers out from a regular gel (tear them out with a pipette tip?).

Alternatively, if you're set on using regular gels for your probing, you could probe with a removable stain like Ponceau S to reveal where your proteins are bound to the PVDF, then mounting it in the Miniblotter and aligning it using the stained areas.  Then you could wash the wells to remove the stain and probe with your antibodies.

One thing I found with the Miniblotter: make sure you have your antibody in block solution.  I always got nasty background on any antibody probings that were simply hybridoma supernatants without adding to 5% BSA (even after blocking the blot).  Not sure if you normally probe in block, but I figured I'd warn you anyway.

Hope that helps.

[hird1026]

Thank you for the reply!!

BTW, when you mentioned:
Alternatively, if you're set on using regular gels for your probing, you could probe with a removable stain like Ponceau S to reveal where your proteins are bound to the PVDF, then mounting it in the Miniblotter and aligning it using the stained areas.  Then you could wash the wells to remove the stain and probe with your antibodies.
I am curious about:
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?

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!!

[John Forsberg]

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).

Quote

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.