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large protein transfer efficiency for western blot
Posted by: biowww (IP Hidden, Regular member, 31)
Date: November 27, 2004 06:28PM
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). I have tried : ... ... Problem with high molecular weight protein western blot [www.bio.net] Comment List -------------------------------------------------------------------------------- Topic: Author: Time: 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 Lu¨ªsa Pissarra 20.02.2004 08:06 I've been having similar problems trying to detect proteins of the 150-180 kDa range. I transfer in a semidry system to nitrocellulose membranes. I've optimized the transfer protocol and now seem to get good transfers which I assess by coomassie staining the gels after transfer (which give very faint bands). I also use a coloured protein marker ladder that transfers with no problem (the 220kDa marker is fainter on the membrane and gets more retained on the gel, but that's quite acceptable since the "cut-off" limit for semidry transfers is around 200kDa). The problem is no matter how good the transfer is I don't seem to get any signal after trying different primary antibodies which seem to work fine in a neighbour lab. We share secondary antibodies and detecting reagents, so they should work fine and I also use about twice the amount of protein extract to start with. Have you any suggestions? RE:Trouble with large protein transfer 02.06.2004 07:55 Hello, I have the same problem for a 160Kda protein DAPK1. I got the transfer ok, but no matter what primary antibody I used, the signal is very very weak. My colleague use the same secondary antibody and other things, and it works fine for them. Have you got any idea of why it didn't work for you now? > I've been having similar problems trying to detect proteins > of the 150-180 kDa range. I transfer in a semidry system to > nitrocellulose membranes. I've optimized the transfer > protocol and now seem to get good transfers which I assess > by coomassie staining the gels after transfer (which give > very faint bands). I also use a coloured protein marker > ladder that > transfers with no problem (the 220kDa marker is fainter on > the membrane and gets more retained on the gel, but that's > quite acceptable since the "cut-off" limit for semidry > transfers is around 200kDa). > The problem is no matter how good the transfer is I don't > seem to get any signal after trying different primary > antibodies which seem to work fine in a neighbour lab. We > share secondary antibodies and detecting reagents, so they > should work fine and I also use about twice the amount of > protein extract to start with. > Have you any suggestions? > 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. RE:Trouble with large protein transfer 16.07.2004 18:56 I am dealing with a protein (200kDa) and it's dimer (400kDa) and also facing problem in transfering them from gel upon the PVDF membrane. I learned from the company that the Tris-Acetate gel system is good for large protein electrophoresis and transblotting. But the problem I have currently is that I am using a BioRad mini- PROTEAN III system and they only offer the pre-casting Tris-Acetate gel for the Midi-Criterion XT. So the only way for me to have the TA gel should be to do it by myself. Does anyone know the gel, running buffer and transblotting buffer formula and be willing to share it? I really need it! Thanks! 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) RE:Having similar problems with transfer efficiency of a 200 08.05.2004 01:57 > Having similar problems with transfer efficiency of a 200 > kDa protien. Referencing "Molecular Cloning, Great Work! Cialis 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)
Re: large protein transfer efficiency for western blot
Posted by: Sasi (IP Hidden, New member, 1)
Date: October 24, 2006 02:33PM
I´m having trouble tranferring fodrin (a 240 KDa) protein. I don´t know which the optimal time and current would be, to transfer it from a 7 and 10% gels on nitrocellulose. I´m using the wet transfer system. I will appreciate your help.
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