What does it mean when an antibody company says an
antibody “does not work” in western blots?
I started wondering about
this question the other day after a colleague told me about a company that
“validates” all of their antibodies by immunostaining in no less than 5
different tissues! We examined the company’s
website and in some cases western blots (WB) showing labeling of a single band
were also presented. But for the vast
majority of products on the site only the apparently innocuous phrase “does not
work in WB” was seen. What does this
mean? I think for many antibody users
(especially people who do not do WB and use various immunostaining protocols
like IF or IHC) this failure to “work” in WB is often interpreted as meaning
something akin to “don’t worry about the WB data, see if the antibody ‘works’
in immunostaining.” However, I think any
user of an antibody that “does not work in WB” should be very worried
indeed. This is because in the
overwhelming majority of cases “does not work in WB” means the antibody labels
many different proteins in a WB. It is
extremely rare to get an antibody that labels nothing in a WB.
Examples of antibodies that do
and do not work in WB are shown in
Fig. 1. The lane at the right shows
staining of a brain lysate with an antibody raised against tyrosine hydroxylase
(THRAB) and it shows labeling of a single band of Mr 60,000 which is the
approximate Mr of TH. The lane at the left
shows staining of an antibody that does not work in WB. The antibody shown was raised against a
protein Sap 102. As can be seen in the
image of the WB, the antibody does recognize a band at the appropriate Mr for Sap
102, but the antibody also recognizes many other bands as well. This antibody ”did not work in WB” and it was
thrown in the trash where it belonged.
So remember when you read
the words “does not work in WB” you should translate that as “this antibody probably
cross reacts with a number of different proteins.”
