can't view the protein band..help! - (Jun/12/2008 )
almost six months doing the same thing..still facing the same problem..protein band can be nicely viewed after ponceus's stain..
so..happily, i continue with probing..start with blocking using 5% Milk-TBST for an hour,tip off, add primer antibody (1:1000,Calpastatin build in mouse),shake at room temperature for 30min,rinse with 1% Milk-TBST for 5 times and 3 min each time then continue with secondary (1:5000, anti-Rabbit IgG peroxidase),shake at room temp for 30min.Rinse again for 5 times and 3 min for each time..tip off.. then I put the ECL reagent (mixed of reagent 1 & 2 in equal volume) on the surface of membrane, and incubate for 1min.. try to view by using gel doc..but no band are visible.. before this, i've checked the reagent, it's work with the same antibody.. FYI the ECL reagent is from Amersham #RPN 2106 and i used semi dry blotting system..i dont know where is going wrong..is it the gel doc system?
almost six months doing the same thing..still facing the same problem..protein band can be nicely viewed after ponceus's stain..
so..happily, i continue with probing..start with blocking using 5% Milk-TBST for an hour,tip off, add primer antibody (1:1000,Calpastatin build in mouse),shake at room temperature for 30min,rinse with 1% Milk-TBST for 5 times and 3 min each time then continue with secondary (1:5000, anti-Rabbit IgG peroxidase),shake at room temp for 30min.Rinse again for 5 times and 3 min for each time..tip off.. then I put the ECL reagent (mixed of reagent 1 & 2 in equal volume) on the surface of membrane, and incubate for 1min.. try to view by using gel doc..but no band are visible.. before this, i've checked the reagent, it's work with the same antibody.. FYI the ECL reagent is from Amersham #RPN 2106 and i used semi dry blotting system..i dont know where is going wrong..is it the gel doc system?
- use PVDF, not nitrocellulose membrane
- reduce time of blocking to 30 min, and use 1% milk.(make sure it is non-fat or use commercial blocking solutions)
- wash briefly with TBST before adding primary antibody
- shake primary for overnight at 4 degrees 1:500
- use 3X10 min wash without milk (only TBST, don't use much Tween 20, it'll rip your proteins and primary off the membrane)
- 1:5000 for secondary is low, make it 1:1000 and use goat anti-mouse as your primary is also mouse
- shake for 1 hour in room temp
- wash without milk 3x10 min with TBST
- add the white and brown ECL of 150 ul each on a flat glass, pipette up and down to mix, lay your membrane on the mixture for 30 sec, ECL will be absorbed, don't worry
- it is not the gel doc....use kodak films with developer and fixer if you want.
good luck