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Semi-dry blotting

Semi-dry blotting - Western Blot Forum

Semi-dry blotting - 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 03-11-2010, 09:09 PM
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Default Semi-dry blotting



I have been having issues with my Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell for about 3 weeks now. We've been blotting 2 - 18 well gels about twice a day. The first issue was that the ladder and proteins, only on one membrane, would bleed, so there was no clear ladder and the proteins were just missing closest to the ladder. I read on one forum for a different problem to try to run one gel at a time. So I figured that would help. Ran about 5 gels this way. The last gel I ran got messed up terribly. The top blotting paper was burnt looking, there was nothing in the gel, and the membrane underneath it had the blue running dye on it. Please someone tell me what's going on!!!
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Old 03-11-2010, 09:54 PM
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Default Re: Semi-dry blotting

More info: Running for 53min @ 20V each time. The burnt looking one was the second of the day. We put the second gel in 4 degrees C while the first gel was blotting. The transfer buffer is 1X tris glycine 15% methanol. And we soak everything for 15 min.
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Old 03-12-2010, 12:12 AM
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Default Re: Semi-dry blotting

I use the same system from Bio-Rad.

Here are my foolproof conditions:

1 gel at a time
Running buffer 1X TGS
Amps set constant = 0.5 (mA or ? forgot the unit)
V = 17V
Time = 19 min

The methanol and extra time are unnecessary in my opinion, you can simply load less protein on gel and get near 100% transfer.

Some quirks of the system.

*Power off the power supply between runs. (use the best power supply in the lab, make sure you are setting Amps constant)
*Monitor Current in Amps, it tells the whole story. If the system quickly gets to 0.4-0.5A then you are doing great. The current/ions are hitting your proteins thus causing the resistance (indicating the transfer is taking place).
On the other hand if you see little resistance (low amps less than 0.3-0.35) then you either don't have good conduct between machine and sandwich, enough buffer, or very low proteins; monitor this for 5-10min if the Amps slowly but surely climb upwards, the problem fixed itself and you'll have a solid transfer.
*Bio-Rad manuals suck and even include major typos, but read up how to setup the sandwich anyway and don't make any mistakes.

Last edited by danfive; 03-12-2010 at 12:47 AM.
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  #4  
Old 03-12-2010, 12:18 AM
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Exclamation Re: Semi-dry blotting

Quote:
Originally Posted by sniftyhit13 View Post
The top blotting paper was burnt looking, there was nothing in the gel, and the membrane underneath it had the blue running dye on it. Please someone tell me what's going on!!!
Don't run the transfer over twenty minutes, waste of time, and you will overheat the sandwich, and have a mess on your hands. Tell me you read the manual, safety first,

BTW in the common tank transfer, 53-60 minutes is OK, but semi-dry will become completely dry, so please control for time. Note that even the tank transfer controls for heat from the system, either by placing tank in 4C or including ice blocks in the tank.

Last edited by danfive; 03-12-2010 at 12:21 AM.
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  #5  
Old 03-12-2010, 12:42 AM
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Post Re: Semi-dry blotting

Here is my protocol written years ago. Most of it is from the Bio-Rad manual, like the guidelines for time and volts on large/small gels. My recommendations are determined empirically.

Western Transfer
Semi-Dry Trans-Blot general instructions
Guidelines:
1.Do not exceed 25V---more will damage the electrodes.
2.Do not adjust pH of transfer buffers—can result in increased conductivity.
3.Only perform short transfer times---can generate too much heat.
4.Use only PowerPac HC/HV-microprocessor controlled low voltage, high current.
Do not use the power supply from Fisher Scientific.
5.Activating the membrane in 100% methanol and then equilibrating the membrane is important—air bubbles can block transfer.
6.Avoid membrane contamination—always use forceps and wear gloves.

Preparation for Blotting:
1.Prepare the transfer buffer.
2.Following electrophoresis, equilibrate the gels in transfer buffer. Equilibration allows the gel to adjust to its final size before transfer. Time depends on gel thickness.
3.Cut membrane to the dimensions of the gel.
4.Wet membrane by sliding it into 100% methanol then equilibrate it in transfer buffer for 15-30 minutes.
5.Cut filter paper to dimensions of gel.
*2 pieces of extra thick filter paper or
*4 pieces of thick filter paper or
*6 pieces of thin filter paper per gel
6. Completely saturate filter paper by soaking in transfer buffer.

Assembly of the Unit:
1.Wear gloves to avoid contamination.
2.Remove the safety cover and stainless steel cathode assembly.
3.Place pre-soaked sheet of extra thick filter paper onto platinum anode. Roll a pipet or test tube over surface to exclude air bubbles.
4.Place the pre-wetted blotting media on top of filter paper. Roll out air bubbles.
5.Carefully place the equilibrated gel on top of the transfer membrane, aligning the gel on center of membrane. Roll out air bubbles.
6.Place the other sheet of pre-soaked filter paper on top of the gel, roll out air bubbles. Saturate the sandwich with transfer buffer.
7.Carefully place the cathode onto the stack Press to engage the latches with the guide posts without disturbing the filter paper stack.
8.Place the safety cover on the unit.
9.Turn on the power supply use the Biorad PowerPac HC not the Fisher Sci. machine.
Mini gels 15-30 minutes 10-15V
Large gels 30-60 minutes 15-25V

Set current limits—to avoid damage due to excess heat.
Mini gels 5.5mA/cm2 Note 0.5A worked on Readygel 17V for 19 min.
Large gels 3mA/cm2
10. Following transfer turn the power supply off, disconnect the unit. Remove the safety cover and cathode assembly. Discard the filter paper. If desired stain the gel to access transfer efficiency.
11. Clean the apparatus with MQ H2O and dry completely before storing.
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  #6  
Old 03-12-2010, 02:19 PM
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Default Re: Semi-dry blotting

Would the gel itself make a difference? We're using 4-12% Bis-Tris 12+2 or 18 well gels. Therefore we're using 1X MOPS running buffer. And I have to say that your protocol is basically exactly mine except we don't watch the amps. I guess this time I will set a limit on the amps.
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Old 03-17-2010, 05:46 PM
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Post Re: Semi-dry blotting

Quote:
Originally Posted by sniftyhit13 View Post
Would the gel itself make a difference? We're using 4-12% Bis-Tris 12+2 or 18 well gels. Therefore we're using 1X MOPS running buffer. And I have to say that your protocol is basically exactly mine except we don't watch the amps. I guess this time I will set a limit on the amps.
Its a must that you control/limit the amps, so that should improve your work.

I looked up my transblot manual for the MOPS running buffer and it is not mentioned at all, the recommended running buffers are Tris-glycine, Tris-glycine-SDS, carbonate buffer.
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Old 03-09-2011, 06:29 PM
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Default Re: Semi-dry blotting

This helped me so much!! Though I don't get a transfer unless I run it for 1:47 mins at 10volts.....
using E-C 150 and the amps hang out around 100.
im baffled
what am i doing wrong?
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  #9  
Old 03-10-2011, 03:37 PM
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Question Re: Semi-dry blotting

Quote:
Originally Posted by mai-mb View Post
This helped me so much!! Though I don't get a transfer unless I run it for 1:47 mins at 10volts.....
using E-C 150 and the amps hang out around 100.
im baffled
what am i doing wrong?
Hi, I'm not sure what you mean.

Do you run 1hour 47 minutes at 10 volts or 1.47min?

What is EC 150?

Is everything completely wet/soaked with tranfer buffer?

Are you sure the membrane is activated and equilibrated with transfer buffer?
(1 min or less in Methanol, 1 minute in MQ H20 while on shaker, 15 minutes or more in tranfer buffer while on platform shaker)

How much protein/lane are you trying to tranfer?
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  #10  
Old 03-10-2011, 07:43 PM
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Default Re: Semi-dry blotting

Yeah I have now done four successful runs,( I'm new at semi-dry).

But it takes an hour and 47mins each time. The volts I cap at 10, Amps plateau out at around 100 (by themselves).

EC-150 is the machine that im hooking the semi dry apparatus up to to gain voltage. Its an ancient thing called thermo EC-150.

I haven't equilibrated in transfer buffer for that long well shaking will do that today!

There is 20ul per lane, or do you mean ug?

Thanks so much, you seem to be very knowledgeable. I'm completely alone in my new lab with equipment Ive never used and no one to ask.
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