Section5:Assay Buffer

From Assay Guidance Wiki

Identify appropriate starting buffer from literature sources or based on experience with similar receptors. Binding assays may require CaCl2, MgCl2, NaCl or other agents added to fully activate the receptor. pH is generally between 7.0 to 7.5. Commonly used buffers include TRIS or HEPES at 25 mM to 100 mM. Protease inhibitors may be required to prevent membrane degradation.

The following are possible factors that can be investigated in a statistically designed experiment to improve radioligand binding to membrane receptors, or reduce radioligand binding to SPA beads. The optimization of the assay buffer may be an iterative process in conjunction with the optimization of the assay conditions to achieve acceptable assay performance. Typical concentrations or concentration ranges for some reagents are listed in the tables below. Other reagents may be required depending on the individual receptor/ligand system.

Note that for most instances, the highest purity reagents should be tested. In some cases, such as with BSA, several forms (fatty acid free, fatty acid containing) may need to be investigated.

Agents which Reduce NSB
BSA	0.05% - 0.3%
Ovalbumin	0.05% - 0.3%
NP-40	0.05% - 0.3%
Triton X-100	0.05% - 0.1%
Gelatin	0.05% - 0.3%
Polyethylenimine	0.01% - 0.1%
CHAPS	0.5%
Tween-20	0.05% - 0.1%
Fetal bovine serum (FBS)	up to 10%

Antioxidants/Reducing Agents
Ascorbic Acid	0.1%
Pargyline	10 μM
DTT	1 mM

Reduce SPA Bead Settling Effects
Glycerol	10 - 20%
Glucose	10 mM
Polyethylene glycol (PEG)	5 - 10%

Divalent Cations
Magnesium (Mg2+)	1 mM - 10 mM
Sodium Acetate	10 mM - 50 mM
Calcium (Ca2+)	1 mM - 10 mM
Zinc (Zn2+)	10 μM - 50 μM

Other Buffer Additives
NaCl	100 mM - 150 mM
KCl	5 mM - 80 mM
TRIS	10 mM - 50 mM
HEPES	5 mM - 100 mM
Phosphate Buffer	20 mM
pH	7.0 - 8.0
Aprotinin	500 units/ml
EDTA	0.51 mM - 5 mM

In addition to Aprotinin and EDTA, other protease inhibitors may be required for receptor stability. As a starting point, Complete™ tablets from Roche Molecular Biochemicals are commonly used.