electrophoresis question - (Apr/17/2007 )
hi there
i am doing IP followed by WB to check my results . i noticed that in some proteins i got 2 bands *one for my IP binding and just close to it another more dense band *i guess its the heavy chain of IgG RIGHT?
SO HOW CAN I GET MY BANDS TO BE MORE SEPARATED FROM THIS HEAVY CHAIN BAND?
I THOUGHT TO PROLONG THE ELECTROPHORESIS TIME FROM 60 TO 70 OR MORE MINTUES *I AM USING 12% gel but my mentor said to try 8% gel for 6 minutes?i dont know why? can you explain?
my band is around 75 by the way.
i am doing IP followed by WB to check my results . i noticed that in some proteins i got 2 bands *one for my IP binding and just close to it another more dense band *i guess its the heavy chain of IgG RIGHT?
SO HOW CAN I GET MY BANDS TO BE MORE SEPARATED FROM THIS HEAVY CHAIN BAND?
I THOUGHT TO PROLONG THE ELECTROPHORESIS TIME FROM 60 TO 70 OR MORE MINTUES *I AM USING 12% gel but my mentor said to try 8% gel for 6 minutes?i dont know why? can you explain?
my band is around 75 by the way.
Hi,
- the other band is bigger or smaller than your protein? Which Ab are you using to IP and which for WB (as in, mouse or rabbit)? It is possible to not detect the heavy chain of IgG with some playing with the 1st and 2nd Abs without need to resort to gel percentage or running time
- Your protein is of average size. I think when you run at lower percentage the bigger proteins (including yours and the IgG heavy chain) could run faster and seperate better.
thankx for your reply
the other band is bigger than my protein and my IP antibody is antirabbit and the first antibody for WB is antimouse.
i dont understand how NOT to detect the heavy chain by playing with antibody? can you explain more?
up?
the other band is bigger than my protein and my IP antibody is antirabbit and the first antibody for WB is antimouse.
i dont understand how NOT to detect the heavy chain by playing with antibody? can you explain more?
Sorry, take me awhile. OK, to not detect the heavy chain, there are at least 2 ways that I know of:
- First: use secondary Ab that would not detect the heavy chain or detect less. In your case, since 1st Ab for WB is mouse, 2nd Ab could be anti-mouse Fab-HRP (check Jackson). This will still detect a bit of heavy chain, but hopefully less. They even have the light chain specific 2nd Ab for WB (that will not detect heavy chain), but it is mouse-anti-rabbit though
- Second, if you don't want to choose the 1st way, then don't use 2nd Ab-HRP. Use Protein A-HRP, Protein G-HRP or A+G- HRP. Mouse antibody should be detected with protein G-HRP, I think.
- Also, some company offer the IP beads that is supposedly able to reduce the elution of antibody, could also help. eBioscience offers TrueBlot Anti-Rabbit Ig IP beads, which does help to reduce the contaminated IgG chains for me.
The main problem with your case is, sine your protein is 70-75kD (right?) and the other band is bigger, I am afraid that it is not the heavy chain. Did you use BSA in any of your buffers?
thank you soo much
and about ur question yes i use 3%BSA in 1x TBST for dilution of my first and second antibody in western blot? does this make a difference?
and about ur question yes i use 3%BSA in 1x TBST for dilution of my first and second antibody in western blot? does this make a difference?
If you don't have BSA in your sample (i.e. lysis buffer) it should not cause the bigger band. The reason why I asked is because in some protocol, the lysis buffer or washing buffer for IP also contains BSA and sometimes FBS. That could cause a big blob at about 80kD when you do WB. But since you did not use it, then it should not be the case. I don't have any idea what cause that extra band then. Your input have that band or it is only in your IP? Can your protein form some complex with other proteins which is resistance to detergents or form some kind of dimer (although for this the size should be bigger)...? Your supervisor is right though, if you cannot eliminate it then run lower percentage gel to get them more separated