How to reduce amount of antibodies used in WB - (Jul/30/2008 )
Hi all!
My problem is the following - usually I incubate my membrane with 5-10 ml of the diluted 1-st antibodies, which is enough to cover the membrane. But this time I need to reduce the amount of the antibody used as much as possible - because the antibody is not so good and I need to use a lot of it. I heard that some people use sealed plastic bags in which they put the membrane and other tricks to incubate comparatively big membranes with small amounts of solution. Maybe somebody can share this techniques.
Thanks a lot!
-Greenrat-
QUOTE (Greenrat @ Jul 30 2008, 06:13 AM)
Hi all!
My problem is the following - usually I incubate my membrane with 5-10 ml of the diluted 1-st antibodies, which is enough to cover the membrane. But this time I need to reduce the amount of the antibody used as much as possible - because the antibody is not so good and I need to use a lot of it. I heard that some people use sealed plastic bags in which they put the membrane and other tricks to incubate comparatively big membranes with small amounts of solution. Maybe somebody can share this techniques.
Thanks a lot!
My problem is the following - usually I incubate my membrane with 5-10 ml of the diluted 1-st antibodies, which is enough to cover the membrane. But this time I need to reduce the amount of the antibody used as much as possible - because the antibody is not so good and I need to use a lot of it. I heard that some people use sealed plastic bags in which they put the membrane and other tricks to incubate comparatively big membranes with small amounts of solution. Maybe somebody can share this techniques.
Thanks a lot!
In my old lab we used to have a Seal-A-Meal device that we used with the plastic bags that were sold with it. Typically one would use 2-3 mls of solution to cover the blot. I've also used petri dishes - about 3 ml/9 cm plate or the glass jars that others use for tissue staining. Alternatively you might want to try to cut off unnecessary parts of your membrane if you know specifically where your protein will run - this could reduce the size of your membrane and how much solution you need to use. You could also try re-using your antibody.
-smu2-
O, I googled seal-a-meal and I think this is exactly what I need. Thanks!
-Greenrat-
QUOTE (Greenrat @ Jul 30 2008, 12:32 PM)
O, I googled seal-a-meal and I think this is exactly what I need. Thanks!
If you decide to use the seal a meal, make certain that you get all of the (largest) bubbles out of your bag. Otherwise the bubbles tend to stay in place over a certain part of your membrane and this can create problems.
-smu2-
QUOTE (Greenrat @ Jul 30 2008, 05:13 AM)
Hi all!
My problem is the following - usually I incubate my membrane with 5-10 ml of the diluted 1-st antibodies, which is enough to cover the membrane. But this time I need to reduce the amount of the antibody used as much as possible - because the antibody is not so good and I need to use a lot of it. I heard that some people use sealed plastic bags in which they put the membrane and other tricks to incubate comparatively big membranes with small amounts of solution. Maybe somebody can share this techniques.
Thanks a lot!
My problem is the following - usually I incubate my membrane with 5-10 ml of the diluted 1-st antibodies, which is enough to cover the membrane. But this time I need to reduce the amount of the antibody used as much as possible - because the antibody is not so good and I need to use a lot of it. I heard that some people use sealed plastic bags in which they put the membrane and other tricks to incubate comparatively big membranes with small amounts of solution. Maybe somebody can share this techniques.
Thanks a lot!
sealed plastic bags as suggested;
you may try to enhance your signal if you have used lower conc of primary antibody by using more sensitive ECL (= more H2O2); another method we tried is a commercial reagent from Pierce, I forgot the name, to enhance the immunosignal; I think they still offer a trial pack (not for free)
-The Bearer-