silly question...maximum loading for 1.5mm gel - (Mar/21/2008 )

again..silly question, what's the maximum protein weight (ug) i can load for the 1.5mm big gel?
I'v done 200ug of total cell, ...seems no big problem for me, but still wondering what's the up limit of that 1.5mm gel?

thanks~~

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theoretically, you can load as much as the slot takes, however, you may like to know which amount is useful; too much protein will not be properly separated; for a 1.5 mm thick mini gel, I think 50 µg per lane is a maximum; do you need it for a Western blot?

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thanks for reply~
I do need it for a western blot.
I'm asking this, because my boss is not so satisfied with my transfer (bio-rad semi-dry transfer, 15v, 45min)
I stained the gel after transfer, there is still a big amount of protein left....
Though the signal from western is decent too....at least for my antibodies, no proteins are lost. (35kd & 17kd)

So i'm thinking...maybe if I reduce the amount of loading, the transfer will be better?~~?

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Membrane has its limit (i mean capacity) to bind proteins, if the amount of protein is too high, most of the proteins will just not bind.

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Nitrocellulose membrane's binding capacity is 80- 100 µg/cm2. And I am loading 200ug of whole cell protein in one well.....
That means I still should be able to get full transfer, right?...

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too much left protein in gel after transfer is not to explain by insufficient binding capacity of membrane; I suppose that there is a problem in transfer which should be optimized; you may offer your precise protocol to open discussion

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Thanks for you all!
Here is my protocol:

10X semi-dry transfer buffer:
58.2g Tris
29.3g Glycine
0.375g SDS
When I use it, I mix 10mL the 10X with 20mL Methanol, and to make a 100mL using water.

Then I submerge the gel(12%), transfer membrane, and blot paper in the 1X transfer buffer for around 10min.
(transfer membrane: Whatman PROTRAN Nitrocellulose transfer membrane;
Schleicher & Schuell Bioscience Gel Blot Paper, 15x20cm)

Then I make a sandwich, trim the paper and membrane, and roll out the bubbles..

And run for 15V for 45min (BioRad Semi-dry transfer cell)

And after 45min, take it out, stain the gel...and I see a lot left over..

Hope I make it clear....

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it is a classical blotting protocol; semidry blotting is not ideal for high Mw proteins or larger amounts; you may increase blotting time, or better use tank blotting

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Thank you~~~

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Transfering high molecular weight proteins with Biorad semi-dry transfert apparatus is difficult. But since your protein of interest is of such low molecular weight, semi-dry transfert should go fine.

As for the transfert, we set the amperes rather than the volts. And we usually set 40 mA per gel for 1 hour. HMW marker do not transfer completely, but 100kDa and under transfer fine.

Have you tried PVDF membranes?

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