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Problems with leaky plates

Problems with leaky plates - Western Blot Forum

Problems with leaky plates - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 02-17-2011, 11:11 PM
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Default Problems with leaky plates



Hi all, I am using Biorad plates, casting frame, and casting stand.

I am seeing leaking no matter what I do. I put the 1mm plate down, with ridged side facing up, the short plate on top of it. They are even, their edges meet in every direction, except the top (which is denoted on the larger plate). I then place into the casting frame, make sure the plates are perfectly flat, lock the mechanism by placing flaps outward, and then I insert into the casting stand, and 'secure' into place. This is, I think, the area where I am going wrong.
Now...if you look at the plates from the bottom, you notice that there is, like from the top, a thin area that water can pass into, or pass out of. Why does water not normally leak from this bottom? Is it because the plates are supposed to be perfectly flat against the bottom of the casting stand? It looks to me that they are, but I still get leakage. I have moved the lever at the top of the casting stand to accommodate the top of the plate, and it 'locks' into place. I put quotations around that because the lock seems relatively insecure, I can pull it out easily if I try.
Ok, that's all I can say for now, thanks in advance for any advice/help.
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Old 02-18-2011, 12:07 AM
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Default Re: Problems with leaky plates

Hi there welcome,
when I used the BioRad plates when I locked the glass into the plastic tray there was NO way I could remove them.

You may either have faulty plastic trays and or locks, may be using the wrong size glass plates, wrong direction of setup.

Note you have to have the shorter side of BOTH of the glass plates (where the wells are face Inwards).

If you still have the manual I would re-read quickly the relevant sections on setup.

I downloaded it and had a quick re-read here:
https://www.bio-rad.com/prd/en/US/ad...d-1ccba156c31c

Click on Documents and download them all and have a quick browse.

Also note the troubleshooting guide, there is 2 types of leaking from the top and the bottom.

If you are getting leaking from the top could be due to:

A: overfill of buffer

Solution for a. Keep buffer level below the chamber leaks chamber overfilled. top of the spacer plate.

B. Improper assembly.

Solution for b. Be sure U-shaped electrode core gasket is clean, free of cuts, and lubricated with buffer. Be sure short plate is under the notch on the gasket, not on top of it.\


EDIT: Sorry I assumed you had leakage post-casting - the user below has good points about casting plate leakage.

Last edited by admin; 02-23-2011 at 11:53 PM.
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Old 02-23-2011, 10:14 PM
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Default Re: Problems with leaky plates

Hi There,

I'm assuming you're getting leaking when you are pouring ('casting') the resolving part of the gel to polymerize...

If so, you are probably getting leaking from the bottom due to the long plate and short plate not being flush on the bottom. To assemble plates, I usually place the green frame on the hard benchtop. I insert both the long and short plates. Then, I slowly slide the entire frame over the benchtop while clamping the plates together. I've found this to be the most efficient way to have the spacer from the long plate even with the short plate. Any gap on the bottom is going to cause your plates to leak.

As an extra precaution, I know others who also use waterproof grease - you smear a thin film along the bottom of the plate before clamping on the casting tray.

Also, wipe off the rubbery sponge on the casting tray everytime you use it to clear any type of debris away. A damp papertowel is sufficient. Then make sure the casting frame is vertical on the frame while clamping.

Finally, your gel should polymerize in 20-40 min, depending on how fresh your TEMED is. If it takes longer than an hour, this will also contribute to a leaking gel.

Hope this helps!
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Old 02-24-2011, 07:57 PM
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Default Re: Problems with leaky plates

Thanks for the help. I ended up just putting a oil/gel resistant piece of material (sponge like) at the bottom of the plates, I don't know if this is always necessary, but it did block leaking, more or less...
my problem continues into a new thread with an eponymous title relative to its error: Too short resolving gel and too long stacking gel
molecularstation.com/forum/western-blot-forum/75281-too-short-resolving-gel-too-long-stacking-gel
since I haven't made enough posts, add an html to the end of my URL.
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