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-   -   Transfer Issues to PVDF (http://www.molecularstation.com/forum/western-blot-forum/4022-transfer-issues-pvdf.html)

Kaitou Kid 09-25-2008 10:31 PM

Transfer Issues to PVDF
 
Hi guys,

I'm new to this forum and I'm using a Biorad transblot machine to transfer the proteins from my gel to the membraben.

I am using PVDF for the membrane. PVDF treatment includes 10-15 seconds soak in methanol and then soaking in transfer buffer for at least 15-20 minutes. I just leave it in there until the SDS-PAGE gel is done.

After that I do the transfer usually at 10 V for 30 minutes, but that hasn't been working so I tried 15 V for 30 minutes today.

After that I stained the gel with coomassie blue for ~2 hours and many bands showed up. The marker was gone and the marker I can see on the PVDF, but the gel is FILLED and I mean FILLED with protein bands.

How can I ensure a complete transfer and is there a way to make sure everything is transferred without having to remake PAGE gels all the time? Thanks guys, I just found this forum and it's great. :notworthy:

:D

admin 09-26-2008 05:32 AM

Re: Transfer Issues to PVDF
 
hello Kaitou welcome to the forums. Thanks for the compliments. hmm 10 V for 30 minutes? that may be your issue.

For 30 minutes you need a very high voltage, I would transfer at a high voltage for about 2 hours to get all the bands off however at 30 minutes I did transfer many times but at much higher voltage than 10 Volts.

Should be about 350 mA constant current for 1 hour or 30 Volts (V) overnight with a stirbar and ice pack in the walk in cooler / cold room.

let me know if you have any more issues or that is not the correct answer i have made many mistakes in western blotting and an expert is one who has made them all :P :)

Kaitou Kid 09-26-2008 03:57 PM

Re: Transfer Issues to PVDF
 
Hi admin, thanks for the input, it's good to know you're a WB expert. I learned a lot from this single WB simply because of the errors I'm running into.

The Biorad semi-dry transfer machine I'm using has a maximum of 25V so I can't go beyond that. The current starts off high around 300+ mA, but it slowly drops down to 85 mA where it stabilizes for the rest of the transfer. I am using a miniGel, which is about 5 cm x 7.5 cm. The manual says to do 10-15V for 30-40 minutes, but obviously this isn't working.

I'm using a semi-dry transfer because my proteins are aroudn 50 kD like actin.

I am going to try and run another one with transfer at 20V for 45 minutes and see what happens. Unfortunately I don't know how to do the transfer in a cold room (no access).

One thing I noticed is that the ladder transferred over to the PVDF, but the proteins did not. Will my extended transfer time result in the ladder going through the nitrocellulose? Is there a way to test if the proteins transfer through the nitrocellulose? Thanks for all your help.

MWRLAB 09-26-2008 09:07 PM

Re: Transfer Issues to PVDF
 
Kaitou,

I had the exact same problem as you. I was also using the Bio-Rad semi-dry.:thumbdown: I tried every combination of voltage and time suggested in the manual with nearly identical results. I solved the problem by switching to the Bio-Rad Mini Trans-Blot Cell and transferring in a 4 degree cold room at 70V for 2 hours or 30V overnight. Both gave me excellent transfer of proteins ranging in size from 10 KDa to 200 KDa. The overnight transfer increased the range to include proteins >200KDa. If you have access to a mini trans-blot system, I would highly recommend it.:thumbs:

danfive 09-27-2008 12:43 AM

Re: Transfer Issues to PVDF
 
Hello people using Bio-rad semi-dry transfer. I've used this for two years and the resulting westerns (in combo with ECL detection) are very high quality and very sensitive, so I'm surprised that you guys are having so much trouble.

One thing is certain is that you won't get most of the protein off the gel, I actually load as little as possible (20-25ug per well, but do 80ug per well when playing it safe).
Next you can call the 800 number for bio-rad and get good help--most of their manuals/instructions are badly written and even have blatant mistakes--I guess that electrophoresis is small potatoes for them.

Anyway, I offer a few suggestions
a) if using nitrocellulose you can check for blow-through by adding a second membrane just behind the first.
b) you can "rinse" after methanol for 15 seconds, by placing the membrane in MilliQ water for 1 minute (I never skip this step) on a shaker if you like, then proceed to soak in transfer buffer.
c) after running, gel can also be rinsed by gently pouring MilliQ water over it (gets excess SDS off), then equilibrated in transfer buffer for about 15 minutes (again I never skip this step).
d) finally for setting up the semi-dry apparatus, make sure you have a very wet setup, but eliminate the bubbles.
e) Most importantly my conditions are limit 0.25 mA, 17 volts, 19 minutes (mini-gel). (Use the amps to set a limit at 0.25mA)
f) only one gel at a time, no matter if two or more fit or manual states otherwise.
g) And always have a good quality (fresh) buffer.

Kaitou Kid 09-27-2008 04:30 AM

Re: Transfer Issues to PVDF
 
Thanks everyone.

Danfive, great tips. I will try them exactly as you say for the next transfer.

One question I have about point E: why is the current set at 0.25 mA? The manual says the current should be between something like 3-5 mA per cm^2 area of the gel/membrane area to be transferred. Of course, you said the manual has mistakes. I found one today in the transblot manual that gives 2 numbers for SDS to add to the buffer. :\

As always thanks a bunch guys. I'll attempt the transfer again tomorrow following danfive's parameters and let you know how it goes.

By the way dan, do you use 10% SDS polyacrylamide gel?

admin 09-27-2008 03:18 PM

Re: Transfer Issues to PVDF
 
Kaitou danfive gave some great points. Also if you are using 10% SDS polyacrylamide gel that may be part of your problem as the higher the percentage the longer/higher voltage you will need to get your proteins out especially the bigger ones.

cheers

Kaitou Kid 09-28-2008 09:39 PM

Re: Transfer Issues to PVDF
 
Hey guys, and update.

My biorad power source can't go down under 10 mA limit, so I set the limit at 290 mA instead following the biorad protocol that say to set the current limit at 5 mA/cm^2 of minigel.

Anyways, I ran TWO GELS, transfer one of them at 15 V for 45 minutes with plenty of transfer buffer.

I stained the first gel that I DID NOT TRANSFER WITH. and the bands were FAINT at best. Stained for 1 hour with coommassie blue.

Then after the transfer I stained the gel that I used to transfer and the bands were VERY CLEAR!!!! There appears to be MORE PROTEIN on the gel I TRANSFERED with...

...what gives?? I am so confused...any help is appreciated. :(

:notworthy::notworthy::notworthy:

danfive 09-29-2008 04:21 AM

Re: Transfer Issues to PVDF
 
Quote:

Originally Posted by Kaitou Kid (Post 9482)
Thanks everyone.



One question I have about point E: why is the current set at 0.25 mA? The manual says the current should be between something like 3-5 mA per cm^2 area of the gel/membrane area to be transferred. Of course, you said the manual has mistakes. I found one today in the transblot manual that gives 2 numbers for SDS to add to the buffer. :

By the way dan, do you use 10% SDS polyacrylamide gel?

Yes it's a mistake, you can call the company to check it out, the experienced support should know about it.
I usually use 4-16% gradient Tris HCL gels and they work fine.

danfive 09-29-2008 04:33 AM

Re: Transfer Issues to PVDF
 
Quote:

Originally Posted by Kaitou Kid (Post 9512)
Hey guys, and update.

My biorad power source can't go down under 10 mA limit, so I set the limit at 290 mA instead following the biorad protocol that say to set the current limit at 5 mA/cm^2 of minigel.

Anyways, I ran TWO GELS, transfer one of them at 15 V for 45 minutes with plenty of transfer buffer.

I stained the first gel that I DID NOT TRANSFER WITH. and the bands were FAINT at best. Stained for 1 hour with coommassie blue.

Then after the transfer I stained the gel that I used to transfer and the bands were VERY CLEAR!!!! There appears to be MORE PROTEIN on the gel I TRANSFERED with...

...what gives?? I am so confused...any help is appreciated. :(

:notworthy::notworthy::notworthy:

Try borrowing a power source or asking anyone about limiting the Amps.

If you are staining only one hour I suggest Imperial Protein Stain from Pierce, not the classic coomassie blue (that's usually an overnight stain with an overnight destain).

I hope you already know the amount of protein you are loading to each well, and perhaps a logical explanation to your different staining patterns is that the transfer gel had its excess SDS washed off (through the procedure) and thus stained more efficiently (that is if you also stained 1 hour etc).


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