Today we're looking at the most basic of blocking reagents: generic protein. I'm talking about the BSA or FCS in your staining (FACS) buffer.
The issue
Non-specific binding of antibodies to cells because antibodies are proteins.
The reagents
BSA or fetal calf serum (FCS, FBS)
Alternatives
Other cheap sources of protein such as milk powder or adult bovine serum. You're better off sticking with the tried-and-true. I favor FCS.
What it’s supposed to do
Prevent non-specific interactions between your antibodies and your cells. More importantly, keep your cells happy (maintain viability) and prevent sticking of the cells to plastic.
Does it work? Yes, although in short staining protocols with happy cells, you may not see much difference. The longer your incubation or the more stressed your cells, the more this will matter.
Examples
Cryopreserved human PBMCs, gated on viable leukocytes:
Note the increased non-specific shift for anti-CD19 StarBright Violet 440 on CD14+ monocytes when staining only in PBS. Neither condition contains any Fc block or any other type of blocking agent, so there's still some non-specific signal we can try to reduce later. The cells here were surface stained for 1hr at room temperature.
Any detrimental effects?
The presence of protein reduces staining with fixable viability dyes. These dyes react covalently with primary amines, which are present to some extent on most proteins. Accordingly, the manufacturer protocols generally recommend washing your cells in PBS and staining in PBS. This isn’t generally necessary. If you use a bit more (~2x) viability dye, you can get perfectly good staining in buffers with standard concentrations of FCS or BSA. Usually the manufacturers recommend using these dyes at concentrations that are ten to fifty-fold too high, so if you haven’t titrated your viability dye down, you’ll probably get better staining (and better viability) if you stain in your standard protein-containing buffer.
How much to use?
Standard amounts are 2-3% serum and 0.5% BSA.
When do you really need it?
Always.
Bottom line/how to get the best results
Include it in your staining buffer. Commercial buffers, such as permeabilization wash buffers, will already contain this.
Reagents used:
Have you observed a difference between different protein concentrations and BSA vs serum?