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[Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

[Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes - Protocols and Methods Forum

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  #1  
Old 03-16-2007, 11:50 AM
WS
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes



Dear experts,

When I incubate two or more membranes with an antibody solution (eg O/
N at 4degC with gentle agitation) and the membranes stick together, in
worst case face to face, will the antibodies still reach their target
proteins and stick there?

In my imagination, the membrane actually has big holes (0.2
micrometers, that's the same one can use for sterile filtration of
antibody solution) that should allow the antibodies to go everywhere,
if there is just enough time for diffusion.

Right or wrong? How much time should one allow to be on the safe side?

What's your suggestion / idea / comment?

Wo

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  #2  
Old 03-16-2007, 03:21 PM
DK
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

In article <1174045841.381085.46510@e1g2000hsg.googlegroups.c om>, "WS" <[Only registered users see links. ]> wrote:

Wrong. "Enough time" is likely to be ridiculously long. In real life, mass
transfer is totally dominated by convection and we strive to reduce
thickness of the diffusional barrier (the non-miscible layer immediate
to the surface) as much as we can - that why we shake, rock, mix, etc.

As an illustration, two destaining gels stuck to each other overnight
will always be visibly blue in the overlap area.

Just incubate blots in the separate trays or sequentially.

DK
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  #3  
Old 03-19-2007, 08:44 AM
newsnet customer
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes



POINT 1:
Incubating the membrane in an antibody solution overnight seems to be overly
long.
The antibody should bind to the protein, but the protein on the membrane may
diffuse into the solution.
Therefore you will get reduced signal on your membrane.
I think 2-3 hours is enough.

POINT 2:
Why would you incubate membranes face to face?
Incubate each membrane separately unless you do not have enough solution.



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  #4  
Old 03-19-2007, 06:12 PM
Kyle Legate
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

newsnet customer wrote:
Are you a troll? You do know that in this newsgroup it's better to give
no information than to give false information, right?

Incubating membranes in antibody overnight is routine, and is in fact
*required* for many antibodies--EGFR antibodies from Cell Signaling, for
example. Also, once proteins are bound to nitrocellulose or PVDF
membranes the interaction is essentially irreversible under normal
antibody incubation conditions. Stripping membranes for reprobing
involves much harsher conditions and proteins still remain bound to the
membrane.

Having said that, incubating two blots in the same vessel is in general
a Bad Idea; avoid it if possible, since regions of overlap will either
have no signal or will be all signal.
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  #5  
Old 03-19-2007, 10:21 PM
Dr Engelbert Buxbaum
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

newsnet customer wrote:


It is, if you do the incubation at 37 degrees (even 1 h is usually
enough). If you do the incubation at 4 degrees, incubation o/n is
appropriate. Advantage of this method: The antibody solution (which
should of course contain an anti-fouling agent like timerosal) can be
re-used many, many times without reduction in signal strength. With
valuable primary antibodies that can be important. I have done an entire
3 year postdoc with a single antibody solution.

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  #6  
Old 03-19-2007, 11:22 PM
DK
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

In article <[Only registered users see links. ]>, Dr Engelbert Buxbaum <[Only registered users see links. ]> wrote:

It's the speed of shaking/rocking that matters more than time at
temperatures > cold room. My rule of thumb: 1 hr at RT with good
rocking or o/n in the cold room. In my hands, the two conditions
give equivalent results.


Another advantage is that after sample prep/gel/transfer/blocking,
it is usually at least 6 pm and one faces a choice between going home
or staying until at least 9 pm to get it the Western finished. Breaking at
the point of primary incubation is both the most logical and safest
point, for the o/n incubation practically gurantees maximum signal at a
given Ab dilution regardless of shaking/rocking speed.

The primary Ab does get diluted and depleted a bit every time you use
it but a factor of two-fold is almost nothing in Westerns so any of it
can be safely disregarded.

DK







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  #7  
Old 03-20-2007, 11:13 AM
newsnet customer
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

>> POINT 1:

Factors that will influence protein binding affinity to membrane:
1. type of membrane.
2. transfer method
3. type of protein.

I have experienced protein diffusion from my membrane after an overnight
antibody incubation. This was confirmed with a positive control. And since
he had no signal or reduced signal on his membrane, this could be a possible
explanation. Please realise that comments on a newsgroup should be taken
with a grain of salt. And you should take a chill pill.

ST





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  #8  
Old 03-20-2007, 12:28 PM
Aawara Chowdhury
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

In <elPLh.13830$[Only registered users see links. ].au>,
newsnet customer <[Only registered users see links. ]> wrote:


So, as Kyle suggested, you are a troll. If your technique in the lab is
as thorough as your knowledge, there are better explanations for your
failed Western.

AC
--
Email: echo 36434455860060025978157675027927670979097959886449 930P | dc
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  #9  
Old 03-20-2007, 09:38 PM
newsnet customer
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Default [Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

> newsnet customer <[Only registered users see links. ]> wrote:

Can you read troll? As confirmed my western failed because of the long
incubation time. However, i'm curious to hear some of your "better
explanations" especially when you don't know the details of my experiment or
the experiment of the original post. Tool.

ST


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  #10  
Old 03-20-2007, 11:10 PM
WS
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Default Diffusion of Antibodies in PVDF and NZ membranes

Stop fighting! It's just about westerns. Not religion, abortion,
soccer and other things people people usually are smashing each
other's heads and become terrorists.

In most cases, at least in my experience, westerns are quite resistant
against anything and the standard procedures as you may find them in
any tech manual or datasheet will do. Of course, they're optimized not
only for good results but rather for good sales, especially when they
suggest you to use a new aliquot of antibody every time.
Naturally, there are special proteins that won't stick well to either
NZ or PVDF or both, so you'll have to do it faster or change the
conditions. I'd recommend changing (i.e. normally increasing) the salt
concentration in the buffers. Adding ethanol or methanol to the buffer
(as you probably do it during the electroblot) also might help, but it
could denature your antibodies and / or kill your detection system. It
must be a pain to find out (how does one do that???) that your
favorite protein does not like the standard conditions. Probably most
people conclude first their prep was bad or that the ab would not
work.

Returning to my initial question, I want to give you some more details
an tell you my experience from the past few days: I'm using westerns
to test for protein phosphorylations in 60 human biopies which are
very very limited samples. I must get most out of it. Two gels were
run and blotted in parallel in order to treat all the samples in the
same way to make the results comparable. O course, they also are
probed with the same ab solution and the ECL is done in parallel. I
now have lots of small stripes (one gel makes six membrane slices
corresponding to different molecular weights) of a PVDF membrane where
always 2 strips go into the same 50 ml tube which contains a primary
AB dilution. By some sort of minimizing of surface principle, these
membrane strips (of course 2 always have the same size) have a
tendency to stick together after some time. One can't control the way
how they do it (face to face, back to back, face to back and vice
versa). Surprisingly (the incubation takes place on a rocker in the
cold room over night), it does not seem to matter much, as I have to
say after several days of analyzing these westerns. Either the
incubation time is long enough to allow for the diffusion of the
antibodies (the package of the membrane says 45um pore size, but I
believe that's a typo, it rather should read 0.45um, but these holes
still should be big enough to let proteins diffuse through) or there
is enough time for the membrane to reach the equilibrium before the
cohesion happens (thus I was lucky) or the cohesion is not strong
enough and loosens from time to time or there is simply enough liquid
and antibody between the sheets.

Best regards,

Wo

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