No bands observed on anti-His western blot - (Jul/31/2008 )
I'm expressing a 30 kDa outermemebrane protein that is targeted to the OM (pE21b vector). I tagged the C-terminus with a 6x his tag for western blot detection. However when I do whole cell lysates I see no bands on the western. I am boiling my cells in 1x SDS-loading buffer for 20 min at 100 deg C before running the gel. I am using the Qiagen petna-his antibody. Ive got this western to work with another protein (same vector, same cloning site) and can see it as a positive control on my western. I'm using a larger dilution then they suggest in the book (Im using 1:12,500), however I can see the positive control. Would it be worth trying more antibody? I've sequenced the gene and can see the his tag in frame. I'm not sure where to go from here any suggestions would be great.
Thanks
do u run any positive control with your protein
1) Try to run the positive control in parallel
2) Try to modify the expression protocol of the protein (temp, time)
3) Apologies, but do you use the correct percentage of gel so it is not running out?
4) Do you see a correct band with Ponceau S or in a Coomassie Gel?
2) Try to modify the expression protocol of the protein (temp, time)
3) Apologies, but do you use the correct percentage of gel so it is not running out?
4) Do you see a correct band with Ponceau S or in a Coomassie Gel?
Yes there is a positive control on the western and it is showing up.
I am running a gradient gel, plus I use the magic marker western blot standard so I can watch the progression of the marker as I run the gel.
The protein is being expressed. I can see it on the coomassie stained gel.
Anyone?
Sometimes tags get masked by folding and are not reachable. You've tried to purify your protein by a using Ni-NTA column?
The last remark seems accurate folding can sometimes hide the tag, it's also possible that you have only a partial expression of your protein (not the entire coding frame) and then the His Tag might not be there I had once this problem 
Did you check accurately your sequence or only the ends ? Is the size of teh protein that you see in Ponceau really coressponding to your full size protein ?
Did you check accurately your sequence or only the ends ? Is the size of teh protein that you see in Ponceau really coressponding to your full size protein ?
Tags can get hidden due to folding, however boiling in SDS for 20min I would expect the tag to be reveled when run on a gel personally.
Did you check accurately your sequence or only the ends ? Is the size of teh protein that you see in Ponceau really coressponding to your full size protein ?
Tags can get hidden due to folding, however boiling in SDS for 20min I would expect the tag to be reveled when run on a gel personally.
I agree with you but theory and bilogy are distinc things believe me
Did you check accurately your sequence or only the ends ? Is the size of teh protein that you see in Ponceau really coressponding to your full size protein ?
Tags can get hidden due to folding, however boiling in SDS for 20min I would expect the tag to be reveled when run on a gel personally.
I boil all my samples in loading buffer (1x) for at least 20 min. This last run I tried boiling in 3x loading buffer for almost an hour with no luck. I also tried 8 M urea, which didn't do anything either.