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Wet Transfer is not transferring 300kDa protein, why?


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#1 klmilum112

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Posted 31 August 2010 - 12:20 PM

For the past 2-3 months, I have been troubleshooting this wet transfer problem I am having. I have been unable to transfer a protein that is roughly 300kDa. I am currently using Invitrogen XCELL Sure Lock mini unit. I usually run a 6% gel with a 5% stacker (I also look for a loading control protein) or an 8% gel with a 5% stacker. I have used nitrocellulose membrane when doing the transfer and it is pre-soaked in the transfer buffer. I'm not sure what is happening and the strange thing is a colleague can get the protein (however, i don't find her results convincing since she sometimes have the same problem too, and is the one who taught me how to use the unit and watched me handle it every time) I have tried the following methods:

Trial 1:

1x Tris-Glycine Transfer buffer w/ 20% MEtOH & 0.4% SDS
250mA (constant), 4hrs, voltage is around 34V but it could've changed
at 4 degrees

Trial 2:

pre-soaked gel in .01% SDS for a minute before setting up sandwich
1x Tris-Glycine Transfer buffer w/ 20% & ).01% SDS (recommended by lifetech ppl)
250mA (constant), 4hrs, voltage (?)
@ 4 degrees

And each time I get something different. Also the samples are run in 40ug, duplicates. It seems that one side of the membrane maybe more intense than the other side, or the 1st sample and the last sample will be more intense. Then there is when I absolutely get no transfer of the high MW protein.

#2 LostintheLab

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Posted 31 August 2010 - 04:32 PM

Have you tried running a different type of gel or staining your gel after transfer to see if those proteins have actually been transferred?
I'm sorry if I'll ask obvious questions, sometimes its these little details that may make a difference.
Are you gel running buffers made fresh everytime? Old buffers can affect the running of gels, maybe the protein isn't getting far enough into the gel for good transfer.
How about your tranfers buffers- fresh too?
And you always carefully remove the air bubbles and press down around the edges?
I'm guessing a 300KD protein will always be at the top of the gels, even a 6% one. I use gradient gels for my 330KD gels- 3-8%, but these little things I find help to get a much better transfer and signal. You could also try semi-dry blotting, I find it works for large proteins for me, although some would say it doesn't work as well, but its always worth a try if you're still having problems.

Just some ideas.
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#3 Roo

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Posted 31 August 2010 - 08:05 PM

I agree with Lost In The Lab, but you might also try pre-equilibrating your gel longer in the transfer buffer (At least 15 min to remove excess SDS in the gel). You can read more about this on the Millipore site (Millipore Westerns.) Read the different subheadings under Factors Affecting Successful Protein Transfer for other ideas.

I know years ago we had some problems with wet transfer that resulted in adding SDS to the transfer buffer, but it was at a very low percentage, 0.05 or 0.1% if I remember correctly. Also, we started adding a stir bar to our wet transfer tank and setting it on top of a stirrer at 4 degrees during the transfer.

Good luck!

Edited by Roo, 31 August 2010 - 08:07 PM.


#4 mdfenko

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Posted 31 August 2010 - 09:28 PM

you should not use more than 0.05% sds in the transfer buffer. it may be required to mobilize the protein but it must be stripped off before the protein binds to the membrane.

have you stained the gel after transfer to see if the protein exited?

if you reuse the transfer buffer then you may not have the methanol concentration that you think you have. you may not be stripping enough sds off of the protein to allow binding.
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#5 klmilum112

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Posted 01 September 2010 - 06:23 AM

@ lostinthelab- Yes I have tried staining the gel with coomassie and the high mw proteins were still there. So I am convince it's not transferring those proteins, but the lower mw proteins are. Buffers are made fresh, every time. I don't re-use buffers. I am positive that I "roll" the bubbles out when I am making the sandwich. Also when you do the semi-dry, what is your current (mA) set at and how long do you run it? The last time I ran a semi-dry unit, I ran it at 120mA (constant, the voltage varied up to 250, and it was for 6.15h. Afterwards, I didn't get the protein at all and I stained it and that's when I saw the high mw proteins on the gel. [I did the semi-dry and wet transfer side by side to compare, both had high MW bands on the gel after staining.]

@ roo -- adding the stir bar helps during the transfer? why? I'm just curious I might try it.

@mdfenko -- Yea, I quit using the buffer with the high sds concentration in it. I just used the one I made that has .01% SDS. The first time I used, I was able to see the protein come out on one side of the membrane, but on the other side, it seemed like the protein "disappeared." Do

#6 LostintheLab

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Posted 01 September 2010 - 05:30 PM

Hi again,

The semi dry I ran at 0.7mA per squared cm of the gel- around 32mA for a 6x8cm gel. I ran for 3hrs for high molecular weight transfer. I'm not sure if that will help you though
How are the sponges for your wet blot? - when they get old they can affect the transfer of proteins.
I also run my wet transfers at room temperature, for 2/3 hrs max. Leaving your blotting machinery at 4C for a long time (I'm speaking of months here) can affect its performance.
I wonder if you ran a lower gel percentage you might get better pentrance in to the gel for the larger molecular weight proteins so they're not so near the edges- might give you better transfer of them then.
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#7 klmilum112

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Posted 02 September 2010 - 06:16 AM

Hi again,

The semi dry I ran at 0.7mA per squared cm of the gel- around 32mA for a 6x8cm gel. I ran for 3hrs for high molecular weight transfer. I'm not sure if that will help you though
How are the sponges for your wet blot? - when they get old they can affect the transfer of proteins.
I also run my wet transfers at room temperature, for 2/3 hrs max. Leaving your blotting machinery at 4C for a long time (I'm speaking of months here) can affect its performance.
I wonder if you ran a lower gel percentage you might get better pentrance in to the gel for the larger molecular weight proteins so they're not so near the edges- might give you better transfer of them then.



HI to You too!

When I was doing the semi-dry, I used an 8 or 8-16% gel, ran it at 20mA for 4hrs and then I had increased it to 6.15h, still got nothing. Probably because the current was to low to transfer that big protein. Now the sponges I have are fairly new. There are some ratty looking ones we have, but I don't use those.
I never tried using the wet transfer at room temp. Do you have to keep it on a bucket of ice or store ice in the side chambers? I'm just thinking about it overheating. But at this point, I'll try anything.
Now yesterday, I transferred with a 6% gel, and used the wet transfer overnight at 250mA. This morning I took the apparatus apart and the markers seemed to transfer (nothing was on the gel). I am just staining the gel now and there are a couple very faint bands that are really high. But I cannot tell what size they are. I am hoping that it's not my protein.

Thanks for your help. You've been very helpful! :)

#8 mdfenko

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Posted 02 September 2010 - 08:08 PM

with high molecular weight proteins you should expect less than complete transfer. if you have very little left in the gel then you did transfer some out of the gel.

did you see the standards on your membrane? if not, are you sure that you have the membrane on the correct side of the sandwich (sorry if this sounds insulting)?
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#9 laurequillo

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Posted 02 September 2010 - 08:14 PM

I am currently working with CBP-GFP (320kda) and I do my semi-dry transfers at 24V (I just fix the V) for 2-3 hours. Gels are 8%, and the transfer is quite nice.
Maybe you could try it

Edited by laurequillo, 02 September 2010 - 08:16 PM.

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#10 klmilum112

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Posted 03 September 2010 - 05:44 AM

with high molecular weight proteins you should expect less than complete transfer. if you have very little left in the gel then you did transfer some out of the gel.

did you see the standards on your membrane? if not, are you sure that you have the membrane on the correct side of the sandwich (sorry if this sounds insulting)?


Yea, the standards were on the membrane (I circle them before I block it) and I'm positive the membrane is on the correct side of the sandwich ( I keep the protocol in front of me).

#11 klmilum112

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Posted 03 September 2010 - 06:01 AM

I am currently working with CBP-GFP (320kda) and I do my semi-dry transfers at 24V (I just fix the V) for 2-3 hours. Gels are 8%, and the transfer is quite nice.
Maybe you could try it


I am going to try that to see if I can get any results. Do you know why it matters if you keep volts constant vs mA? I'm just curious.

#12 mdfenko

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Posted 03 September 2010 - 07:57 PM

Do you know why it matters if you keep volts constant vs mA? I'm just curious.


maintaining voltage constant will cause current to drop and may prevent excessive heating (i use constant current with semi-dry transfer).

holding current constant will cause voltage to increase during the run and will cause increased heating.

Edited by mdfenko, 03 September 2010 - 08:03 PM.

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