large protein transfer efficiency for western blot

A newsgroup post on large protein transfer efficiency for western blot.

I have been having a lot of problems with efficiency of transfer from
gel (10% acrylamide/bis) to PVDF membrane (Immobilon P). When I stain
the gel after transfer, there is a lot of protein left on there. I know
that I am not exceeding the binding capacity of the membrane (I did a
titration of ug protein loaded and discovered that the ratio of protein
transferred to those that stay behind is the same).
The protein of interest is high moleculat weight (around 160
kDa).

Biowww.net discussion threads:

Trouble with large protein transfer
27.05.2003 08:58
Hi there.

I have experienced some problems as well with large protein transfers. I am currently performing blots on the EGF receptor (~180 kDa) and can't seem to get a strong signal. Although I have not stained the gel to see if the transfer was complete, I do use a coloured protein marker ladder that transfers no problem, even the high molecular weight marker (175 kDa).

I have searched how others accomplished effective blots, and most of these authors had immuno-precipitated the EGFR before blotting, whereas I have blotted straight from a lysate. Anyone have some suggestions? I'm thinking of trying SDS in the Transfer Buffer.
RE:Trouble with large protein transfer
07.10.2003 18:44
Use gradient gel (4-15% or 4-20%), add 0.1% SDS in your transfer buffer and lower methnaol concentration in your transfer buffer if you use PVDF membrane!
> Hi there.
>
> I have experienced some problems as well with large protein
> transfers. I am currently performing blots on the EGF
> receptor (~180 kDa) and can't seem to get a strong signal.
> Although I have not stained the gel to see if the transfer
> was complete, I do use a coloured protein marker ladder that
> transfers no problem, even the high molecular weight marker
> (175 kDa).
>
> I have searched how others accomplished effective blots, and
> most of these authors had immuno-precipitated the EGFR
> before blotting, whereas I have blotted straight from a
> lysate. Anyone have some suggestions? I'm thinking of trying
> SDS in the Transfer Buffer.
RE:Trouble with large protein transfer
07.10.2003 18:44
Use gradient gel (4-15% or 4-20%), add 0.1% SDS in your transfer buffer and lower methnaol concentration in your transfer buffer if you use PVDF membrane!
> Hi there.
>
> I have experienced some problems as well with large protein
> transfers. I am currently performing blots on the EGF
> receptor (~180 kDa) and can't seem to get a strong signal.
> Although I have not stained the gel to see if the transfer
> was complete, I do use a coloured protein marker ladder that
> transfers no problem, even the high molecular weight marker
> (175 kDa).
>
> I have searched how others accomplished effective blots, and
> most of these authors had immuno-precipitated the EGFR
> before blotting, whereas I have blotted straight from a
> lysate. Anyone have some suggestions? I'm thinking of trying
> SDS in the Transfer Buffer.
RE:Trouble with large protein transfer
07.10.2003 18:44
Use gradient gel (4-15% or 4-20%), add 0.1% SDS in your transfer buffer and lower methnaol concentration in your transfer buffer if you use PVDF membrane!
> Hi there.
>
> I have experienced some problems as well with large protein
> transfers. I am currently performing blots on the EGF
> receptor (~180 kDa) and can't seem to get a strong signal.
> Although I have not stained the gel to see if the transfer
> was complete, I do use a coloured protein marker ladder that
> transfers no problem, even the high molecular weight marker
> (175 kDa).
>
> I have searched how others accomplished effective blots, and
> most of these authors had immuno-precipitated the EGFR
> before blotting, whereas I have blotted straight from a
> lysate. Anyone have some suggestions? I'm thinking of trying
> SDS in the Transfer Buffer.
similar problem of transferring a protein of high mol wt
04.08.2002 15:55
hi
I am also facing a similar problem of transferring a protein of high mol wt.
I am working on 120 KD protein. I hope the previous comments will help. Apart form that I am having a fair bit of background with it.......... any suggestions

Dr. hema raina

2002-07-11 20:20:01
hemaraina (hemaraina@hotmail.com)
RE:similar problem of transferring a protein of high mol wt
06.12.2002 00:08
> hi
> are you the srinagar wali dhanno!get in touch
whether the protein will react in the same manner
04.08.2002 15:54
hi,
I just wonder whether the protein will react in the same manner while it is in solution and in the gel. I quantified the protein by bradford and based on the result i loaded equal amount of protein in the wells of 14% acrylamide gel, but the gell pattern does not look like that i have loaded equal amount of protein. Do u have any comment for that?
james

2002-03-08 13:47:25
james (laddie81@yahoo.com)

facing a similar problem
04.08.2002 15:49
i too am facing a similar problem of transfering a big protein of 172 Kd in wet transfer buffer overnight at 4 deg at 100mA. i satined the gel after tranfer and foung my protein was still on the gel. my transfer buffer is Tris Base SDS and not glycine.

2001-09-06 06:16:00
Dr. renu walia (dc22@rz.uni-karlsruhe.de)
RE:facing a similar problem
29.11.2002 13:48
My suggestions:

1: Don't use PVDF. I find Nitrocellulose membrane much better.
2: Use Tris-glycine based buffers.
3: Transfer overnight at 100 v.
4: Use 2 pieces of Wattman for each part of the "sandwich".

Apart from this I am stumped.

RE:facing a similar problem
12.09.2003 14:03
> As below, but transfer overnight at 4C and at 30V



My suggestions:
>
> 1: Don't use PVDF. I find Nitrocellulose membrane much
> better.
> 2: Use Tris-glycine based buffers.
> 3: Transfer overnight at 100 v.
> 4: Use 2 pieces of Wattman for each part of the
> "sandwich".
>
> Apart from this I am stumped.
>
>
RE:facing a similar problem
04.08.2002 15:51
I'm working with a 113 kd protein and I have had no problem. I use a 10% gel too. I add 2ml of 10% SDS to 2L of transfer buffer which is Tris based.

2001-10-03 20:05:19
V (arby@hotmail.com)
Having similar problems with transfer efficiency of a 200 kD
04.08.2002 15:48
Having similar problems with transfer efficiency of a 200 kDa protien. Referencing "Molecular Cloning, A laboratory Manuel " (Sambrook, Fritsch, Maniatis) They suggest an effective range of separation of SDS-Polyacrylamide Gels as follows:

15% = 12-43
10% = 16-68
7.5% = 36-94
5% = 57-212

They also recommend the following Transfer Buffer:
39 mM Glycine
48 mM Tris Base
0.037% SDS (electrophoresis grade)
20% Methanol

They have the transfer running with a current of 0.65 mA/sq. cm. of gel for a period of 1.5 to 2 hours.

In several experiments I have seen better transfer of higher molecular weight proteins with the addition of SDS. Also we have had problems with poor grades of Tris base that give acidic pH once dissolved in water instead of the usual ~8.5.
Another aspect that they repeatidly point to is the possiblity of a short by using nitrocelluse or stack paper that is larger than the gel itself. The make a big deal about having these exaclty the same size as the gel.

Hope this is informative. Let me know if it works for you.


2001-06-22 14:21:29
SwingDr (tdschr0@pop.uky.edu)

Last update 20-Dec-2005, Rating Good of 7 votes.