Western Blotting Current Problem
Aaron Polesky
I am having problems with Western blots and need some help. Using
standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
apparatus, I am finding that at constant voltage the current drops
throughout the procedure, starting low at around 120 mA and bottoming out
near 90 mA. I am seeing no successful transfer and would appreciate any
help anyone can offer. Thanks in advance.
--
Aaron Polesky Dept. of Zoology
University of Toronto e-mail: apol...@credit.erin.utoronto.ca
"It's better for us if you don't understand..." - TTH
Think Green...Save the Whale.
Sean Sweeney
In article <4m8mk9$c...@tuzo.erin>, apol...@erin.utoronto.ca (Aaron
Polesky) wrote:
I always do my transfer at constant amps, 150mAmps for 2hours. Transfer
buffer is standard: 25mM Tris, 190mM glycine and 20% methanol (2.9g, 14.5g
and 200ml per litre for each reagent respectively) and I transfer on to
0.2 or 0.45 micron nitrocellulose. Do not pH the transfer solution, it'll
be fine as is. I have never had any problems with this procedure, in fact
I find it almost idiot proof using these conditions (i.e. even I can do
it, therefor it is idiot proof). I always check for transfer by staining
with Ponceau S.
hope this helps
Sean Sweeney
Dept of Genetics
University of Cambridge
Aaron Polesky
I am currently having western blot problems and would appreciate any
advice. I am blotting with the Bio-Rad miniblot apparatus using the
recommended buffer (Tris-glycine-methanol). With fresh buffer at
constant voltage, the current begins at around 120 mA and then drops
quickly to around 90 mA. As the icepack melts the current comes back up
to around 100 mA. As a result of this I'm seeing no transfer. Is anyone
familiar with this problem? Any suggestions? Please respond directly to
me as I don't read these newsgroups regularly. Thanks in advance.
Michael W. Thompson
apol...@erin.utoronto.ca (Aaron Polesky) wrote:
>I am having problems with Western blots and need some help. Using
>standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
>apparatus, I am finding that at constant voltage the current drops
>throughout the procedure, starting low at around 120 mA and bottoming out
>near 90 mA. I am seeing no successful transfer and would appreciate any
>help anyone can offer. Thanks in advance.
With most power supplies, if you turn both knobs (voltage and current) all the way
down, then turn the voltage all the way up and adjust your current with the current knob,
you should not have this problem
Ron Tate
Sean Sweeney wrote:
>
> In article <4m8mk9$c...@tuzo.erin>, apol...@erin.utoronto.ca (Aaron
> Polesky) wrote:
>
> > I am having problems with Western blots and need some help. Using
> > standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
> > apparatus, I am finding that at constant voltage the current drops
> > throughout the procedure, starting low at around 120 mA and bottoming out
> > near 90 mA. I am seeing no successful transfer and would appreciate any
> > help anyone can offer. Thanks in advance.
> I always check for transfer by staining
> with Ponceau S.
>
> hope this helps
>
> Sean Sweeney
> Dept of Genetics
> University of Cambridge
It indeed may be pH. While I don't have any other advice as to the
current drop and failure to transfer problem I do have a suggestion for
monitoring transfer, load a lane with prestained standards, I use
Bio-Rads multicolored ones, at a glance I can tell how well the transfer
went.
--
>>>>>>--------------------------->
>Ron Tate
>Lab of Franklin Leach
>Dept. of Biochem. & Molecular Biology
>Oklahoma State University rt...@bmb-fs1.biochem.okstate.edu
(405) 744-9326
---------------------------------------------
Keld Sorensen
[snip a lot of stuff]
>Any suggestions? Please respond directly to
>me as I don't read these newsgroups regularly.
I don't want to pick on the poster, but is it reasonable
to expect the readers to answer when the poster clearly will
never be able to repay the favor??
Keld.
David Ward Rusnak
> I am having problems with Western blots and need some help. Using
> standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
> apparatus, I am finding that at constant voltage the current drops
> throughout the procedure, starting low at around 120 mA and bottoming out
> near 90 mA. I am seeing no successful transfer and would appreciate any
> help anyone can offer. Thanks in advance.
> --
> Aaron Polesky Dept. of Zoology
> University of Toronto e-mail: apol...@credit.erin.utoronto.ca
> "It's better for us if you don't understand..." - TTH
> Think Green...Save the Whale.
Try constant current 250mA with ice in the insert for cooling. Run for
about 1 to 1.5 hours
No SDS, 20% methanol, 1X tris/glycine.
Nelson said this will blast it out, no problem
Later,
Rusnak and Rhodes
Ralf Breuker
Dear Mr. Polesky,
you wrote:
>
> I am having problems with Western blots and need some help. Using
> standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
> apparatus, I am finding that at constant voltage the current drops
> throughout the procedure, starting low at around 120 mA and bottoming out
> near 90 mA. I am seeing no successful transfer and would appreciate any
> help anyone can offer. Thanks in advance.The standard protocol for semidry western blot according to Khyse-Anderson uses a
constant current of 0,8 mA/sqcm. It is recommodated to use constant current to gain a
predictable migration of proteins independent of gel thickness or number of blot
units. (You may pile several units of blotting paper, membrane and gel seperated by
dialysis membranes).
If constant current leads to no better results, look for airbubbles between the
filter paper or in case of high (> 150 kD) MW try buffer without methanol. (I hope,
you didn't fix the gels, as for standard protein detection?)
Don't hesitate to mail me in case of further problems.
Bye
Thomas Thekkumkara
On 2 May 1996, Michael W. Thompson wrote:
> apol...@erin.utoronto.ca (Aaron Polesky) wrote:
> >I am having problems with Western blots and need some help. Using
> >standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
> >apparatus, I am finding that at constant voltage the current drops
> >throughout the procedure, starting low at around 120 mA and bottoming out
> >near 90 mA. I am seeing no successful transfer and would appreciate any
> >help anyone can offer. Thanks in advance.
>
> With most power supplies, if you turn both knobs (voltage and current) all the way
> down, then turn the voltage all the way up and adjust your current with the current knob,
> you should not have this problem
>
>
> Thomas Thekkumkara wrote:
I believe that is normal. If you transfer proteins at constant
voltage, you will see the Amp going down. You should do the other way.
Transfer your protein at constant Amp. Sometimes it is difficult to do
using regular power supply. You may need a power supply similar to EC570
(E-C Apparatus corporation), which has the capability of high Amp output.
Good luck.
Michael Daws
I had a similar problem with the biorad apparatus. I am now using a
semi-dry blotter - it's cleaner , it's faster, and it's more effective.
My advice would be to get one or find someone who has one and use this -
it's much less hassle
Mike Daws
julian catmull
In article <rusnak~dw-020596...@us4j03.glaxo.com>,
rusnak~d...@glaxo.com (David Ward Rusnak) wrote:
> In article <4m8mk9$c...@tuzo.erin>, apol...@erin.utoronto.ca (Aaron
> Polesky) wrote:
>
> > I am having problems with Western blots and need some help. Using
> > standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
> > apparatus, I am finding that at constant voltage the current drops
> > throughout the procedure, starting low at around 120 mA and bottoming out
> > near 90 mA. I am seeing no successful transfer and would appreciate any
> > help anyone can offer. Thanks in advance.
> > --
> > Aaron Polesky Dept. of Zoology
> > University of Toronto e-mail: apol...@credit.erin.utoronto.ca
> > "It's better for us if you don't understand..." - TTH
> > Think Green...Save the Whale.
>
> Try constant current 250mA with ice in the insert for cooling. Run for
> about 1 to 1.5 hours
>
> No SDS, 20% methanol, 1X tris/glycine.
>
> Nelson said this will blast it out, no problem
>
> Later,
>
> Rusnak and Rhodes
-----------------------------------------------------------
I use the same apparatus without any transfer problems with a constant
voltage of 80V for no more then 1 hour. The mA will drop due to the
temperature increasing which cant be helped. However, you should start
with precooled buffer (your buffer is fine) and the frozen ice pak for the
miniblot, making sure the stirrer is at a high speed. You can include low
concns of SDS (0.1%) to ensure uniform negative charge on all proteins and
to enhance their transfer and lessen the transfer time. However, I have
heard of some strange instances where a protein transsfers in the other
direction so try putting the nitrocellulose membrane on both sides and
probe both. Remember not to over do the transfer because you may be
forcing the proteins out and past the NC. One other thing is that you may
be getting adequate transfer but your blocker is interfering witht he Ab,
this happens frequently with caesin and milk powder (0.4% and 1% resp.).
so also try a different blocker. Finally make sure you use high grade
methanol, nothing less will do.
J. CAT. downunder.
Salia Gherras
In article (Dans l'article) <rusnak~dw-020596...@us4j03.glaxo.com>,
rusnak~d...@glaxo.com (David Ward Rusnak) wrote (écrivait) :
> In article <4m8mk9$c...@tuzo.erin>, apol...@erin.utoronto.ca (Aaron
> Polesky) wrote:
>
> > I am having problems with Western blots and need some help. Using
> > standard transfer buffer (Tris-glycine-methanol) in the Bio-Rad miniblot
> > apparatus, I am finding that at constant voltage the current drops
> > throughout the procedure, starting low at around 120 mA and bottoming out
> > near 90 mA. I am seeing no successful transfer and would appreciate any
> > help anyone can offer. Thanks in advance.
> > --
> > Aaron Polesky Dept. of Zoology
> > University of Toronto e-mail: apol...@credit.erin.utoronto.ca
> > "It's better for us if you don't understand..." - TTH
> > Think Green...Save the Whale.
>
> Try constant current 250mA with ice in the insert for cooling. Run for
> about 1 to 1.5 hours
>
> No SDS, 20% methanol, 1X tris/glycine.
>
> Nelson said this will blast it out, no problem
>
> Later,
>
> Rusnak and Rhodes
You must put a magnetic barrel for mixing your solution and to diminue the
heating by using a nice cooling unit ( noticed biorad ) and working at
100v constant for one hour.
good luck