problem with western blot - (Jan/23/2008 )

hello..i have some problem with my western blot..
i've been doing this twice but still couldn't get the result..i'm using the chemiluminescent method to detect my recombinant protein on the film..i'm using anti-his antibodies conjugated horseradish peroxidase from qiagen as my prob to detect the present of my recombinant 6xhistag protein.but it doesn't show any expression
could somebody help me to troubleshoot my problem..

and i have another question to ask for the help..if there were some of the bases mismatch with the database sequence involving the different varieties of my plant is it effect the whole protein of mine?or could it be different varieties might have a little bit different in their sequence??

if i want to do a colony blot for early detection, do i have to use the freshly transformed colonies after ligation?or after i know my positive colony that bring my insert after i send it for sequencing? this is becoz i've been induce my protein many time but after i run on the SDS-PAGE there were nothing much different between my positive colony and control which is vector without insert.is it becoz my protein are not being express?

i'm really new in protein work, so i have a little bit blur in some cases..so i hope that anyone in this forum would kindly help me..thank you.

[quote name='belle' date='Jan 23 2008, 08:01 AM' post='123578']
hello..i have some problem with my western blot..
i've been doing this twice but still couldn't get the result..i'm using the chemiluminescent method to detect my recombinant protein on the film..i'm using anti-his antibodies conjugated horseradish peroxidase from qiagen as my prob to detect the present of my recombinant 6xhistag protein.but it doesn't show any expression
could somebody help me to troubleshoot my problem..


Troubleshooting Western blots:

Did your transfer work? If you use pre-stained molecular weight ladder you can see this transfer to the membrane. Also briefly stain your membrane with ponceau S before probing to see if your protein has transferred (although make sure you wash it out fully before applying antibodies)

Alter blocking conditions and incubation times - I usually use 5% milk powder in TBST but you can increase/decrease the conc to increase/decrease the stringency of antibody binding.

Alter antibody concentration and incubation times - most should work around 1/1000 dilution but you could go as low as at least 1/250. You can incubate your antibody with the membrane from anywhere to 1 hour to overnight at 4 degrees.

Are your chemiluminescent chemicals still active? They do go off over time, if you make your own make it fresh each time.

Finally are you sure your antibody is still active? I have had problems with some antibodies in that they work fine one week and then the next they stop! Do you have a His-tagged protein you know is recognised by the antibody that can use as a positive control?

Aaaahhh, the wonderful world of Western blotting!!
P

yups..my protein transfer work well..i can see the pre-stained marker on it and i use to stain my gel after that and there were no protein at all on the gel..

i make an antibody dilution 1/2000 (as recommended by the company 1/1000-1/2000).for the blocking reagent i use the blocking reagent that they supply together with the antibodies from qiagen.the problem is, i don't have any positive control to detect 6xhistag recombinant protein, so i don't know whether the antibodies work or not..the antibodies has been 1 month with me after delivery..i really get confused becoz they said that this antibodies (tetra.histag) really good and can detect even minimal exposure of my histag or hidden histag.but i get nothing with it..

one thing, how long usually you expose your membrane on film??i used to expose it 2hours before this...

owh~ ihave to know whether my protein being express or not before i proceed it to purification step..

With our chemiluminescent chemicals we only need to expose the film for upto a minute or two, if you see no signal after 20 minutes theres probably not a lot of point exposing for longer as the chemicals go off. You can check that the chemiluminscent chemicals are working by letting a 1ul drop of your antibody dry on your membrane and then incubating with the chemicals and exposing to film (this is a modification of a dot-blot),

If the chemicals work then at least thats one of the potential problem ticked off your list!
P