What is the equation to calculate the voltage and time for transferring proteins from a gel to the membrane in the western blot technique?

hello every body!

I am transferring proteins on a 45 um NC membrane. when working with 90 volts for 100 mins, complete transfer in not achieved, as bands can be seen on the gel when stained with coomassie blue. is it normal? I load 30 ug of protein per well( 100 volts for 20 mins and then 130 volts for 70 mins).

when working with the same voltage for 150 mins, it looks like that proteins just go through the membrane as I can see the stained ladder on both sides of the membrane and no signal on the membrane. I'm confused. Isn't it too high for transferring protein for 2 hours on 90 volts?

your wise answers will be appreciated.

Initial transfer performed overnight (16hr) at 30V failed because small and mid sized proteins (smaller than 100kDa) transferred right through the membrane (as evident from Ponceau staining of the blot and Coomassie staining of the gel). Transfer buffer used was Bjerrum Schafer-Nielsen buffer (48 mM Tris, 39 mM glycine, pH 9.2, containing 20% methanol) containing 0.1% SDS. Apparatus used is BioRad Mini-Transblot (tank/wet transfer method). The electrophoresed gel, membrane and filter pads were equilibrated in transfer buffer around 30mins prior to transfer.

Do you think two hours perfect time for transfer or you may lose the protein if you exceed?

I have a gel that is 5cm by 8cm and am not sure how to figure out the amount of Amps I need and for how long I should run the dry blotter transfer. We are isolating Lactate Dehydrogenase (LDH) and are running two identical gels (one to be stained with Coomassie blue stain and the other to be used in transferring my gel onto a solid surface through Western Blotting. This is my first time making these calculations and I am a bit nervous what I have might be wrong.

We Are trying to obtain efficient and reproducible western transfer of a 340 kDa protein. We routinely do westerns for proteins of 140 kDa and below with no problems. Does anybody out there have any suggestions for our 340 kDa protein?

Hello,

can someone help me or guide me through BCA assay, what formula is used in the end to calculate the protein concentration.

hi

im a newbie when it comes to lab techniques. i have ttried detecting western blot expression of some proteins but i couldnt get any expression (even housekeeping genes). 

currently in the process of reviewing each step in SDS/Western blot. Could you recommend a fail proof or a tried and tested voltage and timing for SDS PAGE and Western blot? i use fixed voltage and 1.5mm SDS gel thickness. I used 100 V for 1hr 30 mins for transfer of protein size 50kd.

could it be that the proteins are not transferred OR there is overtransfer such that proteins went right through membrane? how should i change Voltage?

I tried to detect these proteins by using 0.45µM nitrocellulose and transfer time was 90 min at 90 V

I am working with 100-120kDa proteins that are expected not to transfer well from SDS-PAGE to a nitrocellulose during semi-dry transfer. However, since our lab doesn't have the wet-transfer cell, I have to get the most of what we have (Bio-Rad Semi-Dry blotter).

After reading various advice on constant amperage vs. constant volts, taking an account for the membrane surface, etc. , I reached to some combination of amperage/time, that gives me "good" transfer. I put the "", because I still have proteins left inside the gel (visualized by commmasie after the transfer), but at the same time I have some molecular weight marker (the one for 150kDa!) that gets onto the filter paper below the membrane.

So has anyone solved the "black box" equation of the semi-dry transfer, using a methodological optimization procedure to determine which are the best settings so that most of the protein of interest is out of the gel, but is on the membrane and is not below the membrane ? Please share.

For me using the molecular weight standard as an indicator of transfer is not relevant, because the total protein amount in 10 microliters of standard is usually about 3-5 micrograms. I was thinking about staining covalently somehow some cheap protein (like BSA) and simply tracking how much die I have left on the gel, on the membrane, and possibly - on a second, back-up membrane. What do you recommend?