14 replies to this topic

[flashboy]

hi all,

i'm currently being frustrated by trying to run my protein through a semi-dry blotting system.... the extracted cell protein (i'm actually looking for UCP-2, 31kDa)  is eletrophoresing very weel (as demonstrated by coomassie) but i haven't managed to get it to transfer yet.

i've tried both continuous and discontinuous buffer systems, and a range of power settings (15V for 30m/1h..... 150mA for 1h.....250mA for 1h....mA per cm2 of gel for 1 h) and none of them have caused protein to show up on ponceau s staining....

i'm using sigma's semi-dry western blotter if that helps

where am i going wrong?

james

Edited by flashboy, 25 March 2004 - 01:22 AM.

[flashboy]

i think i've worked it out, the membrane i'm using is almost 18 months old....... the sheet that comes with it says it'll last 6 months. does this sound right?

[flashboy]

still no joy, have a new membrane and still no transfer!!! can anyone else send me the conditions they use so i have a reference point [trying to transfer a 37kDa protein from a 12% agarose gel to a nitrocellulose membrane]

regards

james

[zhglu]

UCP-2 is a highly basic proteins, so it should be transfered easily and quickly. check your semi-dry system to see whether other proteins (just like marker) are transfered. also check ponsinus staining of your protein, this can be done by dot the purified protein on membrane then check. if it doesn't work, you can use fast green. also, put two membranes on both sides of the gel during transfering, and stain the gel after.

good luck

[WesternBlotter]

Hi James

I can offer you a solution to your problem.

Give me a call.

Best regards
Andrew
07815183742

[oddie]

hi all,

i'm currently being frustrated by trying to run my protein through a semi-dry blotting system.... the extracted cell protein (i'm actually looking for UCP-2, 31kDa) is eletrophoresing very weel (as demonstrated by coomassie) but i haven't managed to get it to transfer yet.

i've tried both continuous and discontinuous buffer systems, and a range of power settings (15V for 30m/1h..... 150mA for 1h.....250mA for 1h....mA per cm2 of gel for 1 h) and none of them have caused protein to show up on ponceau s staining....

i'm using sigma's semi-dry western blotter if that helps

where am i going wrong?

Flashboy, what kind of membrane did you used? PVDF or Nitrocellulose?
I guess there should be no problem with the membrane even if it was 18 months old. After you found that no protein was transferred to membrane, did you coomassie stain gel for the proteins? If proteins were on the gel, I assume that you forgot to add methanol to your transfer buffer. Anyway, try this book "Protein methods 2nd edition". It may help. Good luck.  

[flashboy]

i'm using a nitrocellulose membrane, and have now managed to partially transfer the protein by adding sds to the transfer buffer, still bot 100% though!!! am running at 5V for 90 minutes

[flashboy]

well, bugger it i've tried a wet blotting unit and its transferred perefectly!!!! that's the last time i try semi-dry blotting!!!

[vsc]

that has happened to me too,somehow,I made fresh transfer buffer and it seemed to work great.what are you activating you membrane with?are u washing of the alcohol completely before putting it on the transfer unit?I usually run it at 150mA for 1.5 hours,that works best for me

hope that helps

[flashboy]

i tried everything with the semi-dry unit, a range of voltages and ampages, different buffers and different times but never got more than 25-50% transfer. now i use a biorad wet blot unit, at 100V for 1 hour in towbin buffer, using a nitrocellulose membrane which is presoaked in towbin. get 100% transfer every time...

[joshua.wilson11]

hello,

Try reducing the time of transfer to approximately 10-15min at 100mA. Reason, protein is fairly small, so it will traverse the membrane if left to transfer for an extended period of time. best of luck .

Joshua

[mabusheh]

hi
I had this problem before, then I used a PVDF (0.45umpore size)
I activate the membrane for 2minutes in 100% methanol, then soak the membrane in transfer buffer for 5-10 minutes, I transfer at 20Volts for 20 minutes, and I always get a good result. But wet transfer is the best specially for 2-D gels.

regards

[lola]

so you basically want to say that you are having problem with the semi dry western condition...i dont think that this is only the cause...is there any other thing which you are hesitant to say...

[almost a doctor]

flashboy, on Aug 31 2004, 05:32 PM, said:

i tried everything with the semi-dry unit, a range of voltages and ampages, different buffers and different times but never got more than 25-50% transfer. now i use a biorad wet blot unit, at 100V for 1 hour in towbin buffer, using a nitrocellulose membrane which is presoaked in towbin. get 100% transfer every time...

hey flashboy, when using your semi-dry system, did you ever get transfer of anything (i.e. makers?)  Is anyone in the lab getting successful transfer with the semi-dry blotter but you?   If the answer to both this questions is no, my bet is your blotter is faulty, and you've been banging your head for the wrong reasons. Happened to me in the past, very very annoyed to waste my time "optimising".  Also, make sure the transfer buffer is fresh, as changes in pH, salt content, and MeOH evaporation will make it not work.  

I can perfectly understand why you dont want to ever use semi-dry again after this, but as I've always got better results with semi-dry I thought it was worth telling you my experience  

[kottila]

Had the same problems with a dry blotter once, the iBlot from Invitrogen. Couldn't get a decent transfer of a phospho-protein no matter how hard I tried and then I tried wet once and it worked perfectly. Haven't touched the iBlot again since that.