western protein ladder - (Mar/21/2006 )
Are there any ladders that will appear on the membrane after you add ecl and take a picture? if not, how do you know what size the bands that appear on your membrane are?
See ECL DualVue Western Blotting Markers.
the X-ray film is translucide after revelation,
if you have any prestained ladder you will be able to report it on the X-ray film if you are careful not to move the membrane after exposure.
IMHO the best markers for this are Invitrogen's MagicMark (XP) Western Protein Standards. These markers have been engineered to carry an IgG epitope and will therefore react with your secondary antibody (if it's against IgG). They will then give of luminescence (or fluorescence) along with your samples. Here's an example below.
I tried the biotinylated marker from Cellsignaling (or New England Biolab). It works great. It covers broad-range sizes and the sizes of the markers are accurate. It is sold as a kit containing the marker and the anti-biotin antibody conjugated with HRP, so you don't need to buy any extra antibody.
It sounds interesting, but what about unspecific binding of the anti-biotin AB? Do you have any problems with that?
I use MagicMark XP Western Standard from Invitrogen. It does require an anti-rabbit-HRP as the secondary but it work wonders.
MagicMark (XP) Standard react with most common anti-IgG antibodies. Rabbit, cow and human happen to be the best and mouse isn't so good but I use anti-mouse-IgG regularly and I still get an adequate signal. To be sure you just add 2 µL instead of 1.
I tried the biotinylated marker from Cellsignaling (or New England Biolab). It works great. It covers broad-range sizes and the sizes of the markers are accurate. It is sold as a kit containing the marker and the anti-biotin antibody conjugated with HRP, so you don't need to buy any extra antibody.
It sounds interesting, but what about unspecific binding of the anti-biotin AB? Do you have any problems with that?
I have used that one...and it always bound to endogenous biotin...so I always had at least one extra band. Have you used this marker with other gel % than the cell signalling picture (10%). I never managed to get nice marker bands with low percentage gels.
I am using MagicMark (Invitrogen) with anti-mouse-IgG and it works perfectly. But it is supposed to work with almost any anti-IgG.