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Why do I have to almost "burn" my transfer before I can detect??


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26 replies to this topic

#1 medchemgirl

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Posted 21 October 2009 - 11:35 AM

Hi,
I am completely puzzled by this. Every time I try to transfer a particular protein, it wont stick to the membrane unless I run it to the point where the buffer evaporates, almost like baking the system. I am using the semi-dry blotting system from Invitrogen. Even if I use the same amount of voltage (50V, Invitrogen protocol calls for 20V) and keep it really cold that it doesnt evaporates, I won't see my protein, unless I let it go until the buffer evaporates and it smells like burnt! I don't get it! is my protein that hard to to stick? its a 17KDa protein, very small!
I have done this multiple times as to determine that the only way I can detect it is by frying the membrane!

Any comments please?? I'm about to call Invitrogen and ask about this.

#2 HomeBrew

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Posted 21 October 2009 - 12:37 PM

What kind of membrane are you using? What is the composition of your transfer buffer? What kind of gel are you transferring from? How are you detecting the protein?

#3 medchemgirl

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Posted 21 October 2009 - 12:40 PM

Sorry,
I am using NT (Biotrace from VWR), I'm using the Invitrogen NuPAGE transfer buffer (10%MetOH), NuPAGE Novex Bis-Tris gel. I'm using HRP as 2ry with ECL detection.

What kind of membrane are you using? What is the composition of your transfer buffer? What kind of gel are you transferring from? How are you detecting the protein?


Edited by medchemgirl, 21 October 2009 - 12:44 PM.


#4 lab rat

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Posted 21 October 2009 - 02:44 PM

I had problems with the NuPAGE systems, so now I use Biorad Tris-HCl gels and make my own transfer buffer:

3.03 g Tris base
200 ml methanol
500 ml H2O
adjust pH to 8.3
add 14.4 g glycine
q.s. to 1 L

Isn't the max voltage for a SD 15V? (I use BioRad.)

Edited by lab rat, 21 October 2009 - 02:45 PM.

42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#5 HomeBrew

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Posted 21 October 2009 - 04:50 PM

We use the NuPage system as well, and the NuPAGE transfer buffer -- we used to make it up with 20% MeOH, but now we use 20% EtOH -- less disposal problems, less toxicity, and it works just as well. The real difference is we use 20% alcohol, whereas you're using 10%. I've heard spiking it with some SDS can help the transfer of difficult proteins, but we haven't had to try it. We're using the XCell SureLock Mini-Cell apparatus with PVDF membranes.

#6 K.B.

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Posted 22 October 2009 - 01:36 AM

I get very nice transfers of low MW proteins (as low as 6-8 kDa) with Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9, 20% MeOH) and Immobilon Psq from Millipore (PVDF, 0.22 um) on semi-dry system for 1 hour at 0,8 mA per cm2 of membrane.

Edited by K.B., 22 October 2009 - 01:36 AM.


#7 medchemgirl

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Posted 22 October 2009 - 07:39 AM

Thanks a lot!
The max voltage for the Invitrogen Xcell SureLock is 20V, but it wasn't working with 20V so I had to crank it up to 50V accidentally evaporating the inner chamber buffer and luckily getting my transfer to work! I've repeated it several times with same results, if I don't do that, everything else transfer except my protein. I proved it with the standard protein as well.

I had problems with the NuPAGE systems, so now I use Biorad Tris-HCl gels and make my own transfer buffer:

3.03 g Tris base
200 ml methanol
500 ml H2O
adjust pH to 8.3
add 14.4 g glycine
q.s. to 1 L

Isn't the max voltage for a SD 15V? (I use BioRad.)



#8 medchemgirl

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Posted 22 October 2009 - 11:19 AM

That Immobilon membrane sounded good until I saw the price!! haha.

I get very nice transfers of low MW proteins (as low as 6-8 kDa) with Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9, 20% MeOH) and Immobilon Psq from Millipore (PVDF, 0.22 um) on semi-dry system for 1 hour at 0,8 mA per cm2 of membrane.



#9 HomeBrew

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Posted 22 October 2009 - 12:16 PM

That Immobilon membrane sounded good until I saw the price!! haha.


We use 0.22 um PVDF membrane/filter paper sandwiches from Invitrogen. With price, it's relative -- if you've done the experiment multiple times, and haven't got the data or image you need, and it would have worked the first time using PVDF, have you really saved anything?

#10 lab rat

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Posted 22 October 2009 - 02:16 PM

"if you've done the experiment multiple times, and haven't got the data or image you need, and it would have worked the first time using PVDF, have you really saved anything?"

Ditto the SD blotter if it gets damaged by too-high voltage.
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#11 medchemgirl

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Posted 11 November 2009 - 01:55 PM

Well, I finally bought Inmobillon Psq, and still the same problem. This time I tried on the semi-wet system (xcellII blot module) 30V for 30 mins as the tech support of Invitrogen suggested and nothing!!!!! The loading marker perfectly transferred though. I'll increase the time to see what happens.

#12 medchemgirl

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Posted 11 November 2009 - 01:56 PM

I will try this buffer next time!!

That Immobilon membrane sounded good until I saw the price!! haha.

I get very nice transfers of low MW proteins (as low as 6-8 kDa) with Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9, 20% MeOH) and Immobilon Psq from Millipore (PVDF, 0.22 um) on semi-dry system for 1 hour at 0,8 mA per cm2 of membrane.



#13 HomeBrew

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Posted 11 November 2009 - 05:07 PM

I suspect it's something other than the transfer...

Do you know you have a significant amount of protein in the gel? Have you tried staining the membrane (if it's there by staining but not by your HRP protocol, then it's your detection method and not the transfer that's screwing you up)? Try putting a piece of membrane on the other side of the gel as well; maybe your protein is oppositely charged at the pH of transfer..

Did the low molecular weight portion of the ladder transfer correctly?

You could also try 20% MeOH in the transfer buffer, and/or adding some extra SDS...

#14 medchemgirl

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Posted 12 November 2009 - 07:41 AM

Well, I haven't stained the gel after SDS, but I have use a standard protein, I load from 5 to 25 ug of pure protein, and I have nice blots with it, only when I let the system dry, otherwise I don't see anything. From my real samples I load 50 to 100ug of sample.

I suspect it's something other than the transfer...

Do you know you have a significant amount of protein in the gel? Have you tried staining the membrane (if it's there by staining but not by your HRP protocol, then it's your detection method and not the transfer that's screwing you up)? Try putting a piece of membrane on the other side of the gel as well; maybe your protein is oppositely charged at the pH of transfer..

Did the low molecular weight portion of the ladder transfer correctly?

You could also try 20% MeOH in the transfer buffer, and/or adding some extra SDS...



#15 medchemgirl

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Posted 12 November 2009 - 02:37 PM

Is that like a 100V? My system uses volts.

I get very nice transfers of low MW proteins (as low as 6-8 kDa) with Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9, 20% MeOH) and Immobilon Psq from Millipore (PVDF, 0.22 um) on semi-dry system for 1 hour at 0,8 mA per cm2 of membrane.


Edited by medchemgirl, 12 November 2009 - 02:38 PM.





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