Technique / Proteomics and protein biochemistry / Western blot assay
Western blot problem
Western blot problem
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 : ... ...
Last update 22-Jun-2001, Rating of 9 votes.
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By Arun
on 04-Feb-2009
I hope you are using Acrylamide:bisacrylamide in the ratio of 29:1, and not only 30%acrylamide alone. |
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By mssh
on 15-Oct-2008
Hi, I am having problems with my film..after my western is done. I am working with NOS in the hippocampus, and when I did my film for 25 minutes I did not get any bands, but when if i stripp the membrane, then I see a band (not as strong though). What are the reasoning's behind this...how come I don't get any results the first time, but I get a band when I stripp? Is it due to antibody or improper amount of antibody-human error? |
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By Sandhya
on 27-Jul-2008
Make sure that you keep buffer level maintaned. Initially I also had the same problem. |
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By AC
on 14-Feb-2008
I am having trouble with my western blot. I use high concentrations of lysate and antibody. However, I am getting blank results. Can anyone tell me what may be the problem? |
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By HongLien
on 23-Sep-2007
I used Western blot to detect MMP-10 in cultured VSMC but did not receive any signal on film, Please advise me. |
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By Fontenele
on 02-May-2007
Mahboubeh, do you have a very well protein extraction in your Lab. In my work some proteins require different extraction protocol (ie. different lysis buffer). |
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By Mahboubeh
on 26-Mar-2007
I face with a problem in western blot.when I transfer proteins to nitrocellulose membrane it can not be detected protein with DAB or ECL solution,could you kindly help me?meanwhile my prestained molecular weight marker is transfered well. |
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By Hem Chandra Jha
on 14-Jul-2005
Hi, I am facing one problem, I don't know about , How much quintity of protein load in SDS -PAGE when I transfer to PVDF membrain. Is this variable to organism to organism. |
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By Timea
on 16-Mar-2005
Hi, I would like to know, how can I quantify my western blot. I dont want to know the real amount of protein, only compare my samples. Can I do it by comparing the intensity of bands on the membrane, wich I have scaned before? |
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By Sana
on 07-Oct-2004
Everything in western blot is working fine EXCEPT my gel/samples. I dont see my front line going down as straight blue line, i see instead blue dots...and my lanes expand horizontally so at the end of western blot, I see connecting lines (impossible to differentiate between lanes..!!)why is that? is my gel technique messed up or is it my sample? or is it my calculation? maybe someone can tell me how they calculate out and prepare there samples for loading..( I use 5xsample buffer...loading buffer and protein sample x....) |
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By azam
on 28-Sep-2004
When I transfer into nitrocellulose paper Pre stain marker does not transfer properly then it is difficult to point out marker position and I have another problem during transfer voltage increase and electricity flow decrease.If any body have experience please suggest me. |
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By Lin Yihui
on 01-Aug-2003
I found that the Histone 3(<15KD)was not easy to transfer to Nitrocellulose,help me! |
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By zhong
on 22-Apr-2003
Hi, I tried to detect EGFR (>170 kDa) on the cell membrane. I know there are EGFR on the membrane since I detected them by confocal microscopy. And the positive control (EGFR purchased from Sigma) can be detected in the western blot. I boiled the cultured cell pellet in SDS buffer for about 5 minutes, but can not detect them. Who can give me some suggestion about detect membrane receptor by western blot. Thanks. |
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By zhong
on 22-Apr-2003
Hi, I tried to detect EGFR (>170 kDa) on the cell membrane. I know there are EGFR on the membrane since I detected them by confocal microscopy. And the positive control (EGFR purchased from Sigma) can be detected in the western blot. I boiled the cultured cell pellet in SDS buffer for about 5 minutes, but can not detect them. Who can give me some suggestion about detect membrane receptor by western blot. Thanks. |
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By janak77
on 16-Apr-2003
hi i tried to seperate my 45 kd protein from kidney. but i didnt get expression. what are the main reasons behind that. i tried everything. my gel was running fine. it transfered well but in final step i didnt get anything?? |
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By janak77
on 16-Apr-2003
hi i tried to seperate my 45 kd protein from kidney. but i didnt get expression. what are the main reasons behind that. i tried everything. my gel was running fine. it transfered well but in final step i didnt get anything?? |
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By congmiao
on 18-Feb-2003
I have the same problem of the poor transfer onto the membrane. I use 7.5% SDS page gel. 190KD band could not be transfered onto the membrane when I stain the gel after transfer. I use . I trsfer buffer is tris, glycine, SDS and methanol buffer. Who could give me some idea. and by the way, if the protein charge influence the transfer ? If the protein supposed to be a negative charged protein, it should be like what... Most of the protein should be nagetive charged... Now I try to find solution ... thanks. |
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By congmiao
on 18-Feb-2003
I have the same problem of the poor transfer onto the membrane. I use 7.5% SDS page gel. 190KD band could not be transfered onto the membrane when I stain the gel after transfer. I use . I trsfer buffer is tris, glycine, SDS and methanol buffer. Who could give me some idea. and by the way, if the protein charge influence the transfer ? If the protein supposed to be a negative charged protein, it should be like what... Most of the protein should be nagetive charged... Now I try to find solution ... thanks. |
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By yingliwu Dr
on 06-Dec-2002
i agree with SwingDr,i work on a protein of 210kd,and it can be transfered efficiently. |
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By nmatta
on 23-Nov-2002
Hi, I´m having problems with the time of running, Im using acrilamida10%, running buffer with (Glycine, SDS, and tris), the total time its about 10 hours. Does anyone can help me |
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By hemaraina
on 11-Jul-2002
hi |
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By james
on 08-Mar-2002
hi, |
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By V
on 03-Oct-2001
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. |
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By Dr. renu walia
on 06-Sep-2001
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. |
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By SwingDr
on 22-Jun-2001
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: |
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