Why 2 bands for housekeeping? - western blot (Jul/24/2007 )
Hi, can anyone offer me some explanation on why 2 bands appear for a housekeeping?
I'm using stimulated RAW264.7 macrophages and have been probing my membranes with anti-actin (Sigma) & anti-GAPDH (Chemicon). Instead of one band, I get 2 bands for both (the 2 bands lie close to each other). Wondering whether the problem lies in my sample or due to incompatible antibody used.
May I know if anyone is using the same cell line, who can suggest me a good antibody for housekeeping control? I have found other type of antibodies, some from different companies, but haven't proceeded to purchase since I'm worried that I will waste my time and money if the antibody doesn't work.
Thanks in advance.
-money-
QUOTE (money @ Jul 24 2007, 08:01 PM)
Hi, can anyone offer me some explanation on why 2 bands appear for a housekeeping?
I'm using stimulated RAW264.7 macrophages and have been probing my membranes with anti-actin (Sigma) & anti-GAPDH (Chemicon). Instead of one band, I get 2 bands for both (the 2 bands lie close to each other). Wondering whether the problem lies in my sample or due to incompatible antibody used.
May I know if anyone is using the same cell line, who can suggest me a good antibody for housekeeping control? I have found other type of antibodies, some from different companies, but haven't proceeded to purchase since I'm worried that I will waste my time and money if the antibody doesn't work.
Thanks in advance.
I'm using stimulated RAW264.7 macrophages and have been probing my membranes with anti-actin (Sigma) & anti-GAPDH (Chemicon). Instead of one band, I get 2 bands for both (the 2 bands lie close to each other). Wondering whether the problem lies in my sample or due to incompatible antibody used.
May I know if anyone is using the same cell line, who can suggest me a good antibody for housekeeping control? I have found other type of antibodies, some from different companies, but haven't proceeded to purchase since I'm worried that I will waste my time and money if the antibody doesn't work.
Thanks in advance.
Actin sometimes gives more than one band - make sure you are using beta-actin and not actin total
-syaniv-
The one I'm using is monoclonal anti beta-actin
-money-