Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Western Blot Forum
Register Search Today's Posts Mark Forums Read

Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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.


Reply
 
LinkBack Thread Tools Display Modes
  #11  
Old 03-11-2011, 03:51 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Post Re: Semi-dry blotting

Quote:
Originally Posted by mai-mb View Post
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.

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.
I think you should do some practice runs (unimportant sample/protein) and optimize. Semi-Dry is supposed to save you a lot of time versus the traditional tank-tranfer (1 hour to Overnight transfer times).

You should set constant amps at 0.5 or therabout (up to .7 should be fine); this plus short time protects the apparatus and proteins from excess heat.

Volts are probably too low, try 19-20V. Warning do this strictly with 20 minute time limit (go up to 25 max) and limit amps. 1 hour+ will burn the sandwich or proteins, even the apparatus (not huge danger, but a waste of your time/resources).

About the proteins 20ul doesn't tell me anything, ug/lane is much more informative. 20 ug and 20 minute transfer is more than enough. In my experience I load 80ug/lane (heterogenous mix of proteins) and 19 minutes at 17V is enough (0.5A max limit) to get 70-85% of the protein transfered (I stain the used gels with coomassie blue, imperial stain from Pierce actually).
With only large size proteins retained, all small proteins, less than 40 or 50kDa, transfer completely.

The main reason for the short time is that for transfer to occur, everything must stay wet with buffer, plus the convenience of saving yourself time. I don't think this will happen at 1 hour + transfer times.

If you can't limit the Amps, then perhaps you will have to keep volts down somewhat (like 12-14V and increase time 25-30 min max).

Last edited by danfive; 03-11-2011 at 03:53 PM.
Reply With Quote
The Following User Says Thank You to danfive For This Useful Post:
mai-mb (03-11-2011)
  #12  
Old 03-11-2011, 07:16 PM
Pipette Filler
Points: 358, Level: 7 Points: 358, Level: 7 Points: 358, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2011
Location: SF
Posts: 19
Thanks: 10
Thanked 5 Times in 5 Posts
Default Re: Semi-dry blotting

Yeah Ive been using very abundant protein we have running run after run.

I'm doing another run today so i'm going to try to do what you suggested 20V with a limit of 1 amps.

Yeah after a run that long I would expect the transfer buffer to be used up,
I use very little simply saturating the buffer sandwich, but after the hour 47mins the sandwich is still quite saturated.

I have a feeling ive made a rookie mistake or this machine is broken and the volts running through are next to nothing. Thanks for all your help, again!!
oh do you heat your samples at 95 or 100?
Reply With Quote
  #13  
Old 03-25-2011, 12:10 AM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: Semi-dry blotting

Quote:
Originally Posted by mai-mb View Post
Yeah after a run that long I would expect the transfer buffer to be used up,
I use very little simply saturating the buffer sandwich, but after the hour 47mins the sandwich is still quite saturated.

...... do you heat your samples at 95 or 100?
WOOW!!, hold it, my sandwiches are bone dry after semi-dry blotting 19-20min as described before. So that is your problem, this is not normal.


You probably have bad contact, perhaps the blot filter papers are not the right thickness, double/triple up and problem solved. Or maybe the little springs in the semi-dry blot apparatus are very compressed and not pushing the platinum anode out enough---just push them out for them to decompress, slowly and gently.

[Could also be buffer just isn't running buffer, no ions to run---check that the buffer makeup is actual running buffer recipe and concentration (proper water source was used).]

Clean the semi-dry apparatus (not the power source) with copious amount of DI water make sure buffer salts aren't beneath the plaforms (both lid and bottom), terminals (where power source wires make contact)---then leave them Upside Down over paper towels overnight to drip to complete dryness. Check for a springiness to the apparatus lid, the platinum coated platform should be bouncy, any flaky stuff and you have salt buffer build-up in there.

__________________________________________________ ___
Yes samples are boiled in Laemmli loading buffer for 5 minutes, then quickly centrifuged before loading on gel.

Remember after the blotting, you can coomassie or Imperial Blue stain the gel to check transfer efficiency.

Last edited by danfive; 03-25-2011 at 12:17 AM.
Reply With Quote
The Following User Says Thank You to danfive For This Useful Post:
mai-mb (06-29-2011)
Reply

Tags
blotting , semi-dry , semidry , western blot


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Norther Blotting & Southern Blotting Subhabrata Dev Molecular Biology Techniques 1 02-26-2008 01:17 PM
western blotting help (leelavati shetty) Tsalavos Sotiris Protein Forum 0 01-08-2007 01:50 PM
scambio di semi ladybat Forum Biologia 10 08-06-2006 01:37 PM
[Alberi] Metodo germoglio semi Rubens Forum Biologia 2 11-30-2005 06:30 PM


All times are GMT. The time now is 11:24 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13580 seconds with 16 queries