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ROBERT F. REILLY
cipitation. Shorter incubation times are also possible since binding is virtually instantaneous. 100 ).11 of packed protein A -Sepharose will bind about 2
mg oflgG. The IgG concentration in immune sera is approximately 10 mg/
ml. Beads with a significant number of charged groups or hydrophobic
spacer arms should be avoided since they are more likely to exhibit high
levels of nonspecific binding.
Washing: Usually 2-3 washes are sufficient. Each wash should dilute the
soluble phase by about 100-fold. Protein A-Sepharose requires very little
centrifugation. Many monoclonal antibodies are oflow affinity, and if antigen is not precipitated the number of washes should be decreased. Alkaline
pH stabilizes the binding of immunoglobulin toprotein A, and the strength
of the interaction is independent of salt concentration over a wide range.
Elution: Resuspend in SDS-PAGE sample buffer, boil, and load.
Radiolabeling the antigen
One must first decide how to radiolabel the antigen. There are three options:
• Iodination
This technique is used for surface antigens. It has the advantage that only
a small percentage of total cell proteins are membrane proteins. Therefore, a considerable enrichment is achieved from the labeling process
itself. Cytoplasmic labeling can occur if dead cells are present in the sample.
• Carbohydrate Labeling
This can be carried out using either metabolic or enzymatic methods
with tritiated mannose, fucose, galactose or glucosamine. Sialic acid cannot be used because it is not taken up by cells. Enrichment is achieved
since glycoproteins make up less than 20% oftotal cell protein. The technique suffers from low specific activity. Analogously, one can also use
sulfation or phosphorylation.
• Metabolie Labeling With Amino Acids
35 S-methionine is the label of choice. The sulfur- containing amino acids
have a high specific activity (500-1000 Ci/mmol), a relatively strong decay, a half-life of 87 days, and a fairly low cost. Methionine and cysteine
are supplied in aqueous solution containing 0.1 o/o ß-mercaptoethanol ( 12
mM). Cells can only talerate 0.8 mM ß-mercaptoethanol or 500 )lCi/ml.
One should avoid repetitive freeze-thawing of the isotope. The tritiated
amino acids are the second choice (20-200 Ci/mmol). The 14 C-amino
ROBERT F. REILLY
cipitation. Shorter incubation times are also possible since binding is virtually instantaneous. 100 ).11 of packed protein A -Sepharose will bind about 2
mg oflgG. The IgG concentration in immune sera is approximately 10 mg/
ml. Beads with a significant number of charged groups or hydrophobic
spacer arms should be avoided since they are more likely to exhibit high
levels of nonspecific binding.
Washing: Usually 2-3 washes are sufficient. Each wash should dilute the
soluble phase by about 100-fold. Protein A-Sepharose requires very little
centrifugation. Many monoclonal antibodies are oflow affinity, and if antigen is not precipitated the number of washes should be decreased. Alkaline
pH stabilizes the binding of immunoglobulin toprotein A, and the strength
of the interaction is independent of salt concentration over a wide range.
Elution: Resuspend in SDS-PAGE sample buffer, boil, and load.
Radiolabeling the antigen
One must first decide how to radiolabel the antigen. There are three options:
• Iodination
This technique is used for surface antigens. It has the advantage that only
a small percentage of total cell proteins are membrane proteins. Therefore, a considerable enrichment is achieved from the labeling process
itself. Cytoplasmic labeling can occur if dead cells are present in the sample.
• Carbohydrate Labeling
This can be carried out using either metabolic or enzymatic methods
with tritiated mannose, fucose, galactose or glucosamine. Sialic acid cannot be used because it is not taken up by cells. Enrichment is achieved
since glycoproteins make up less than 20% oftotal cell protein. The technique suffers from low specific activity. Analogously, one can also use
sulfation or phosphorylation.
• Metabolie Labeling With Amino Acids
35 S-methionine is the label of choice. The sulfur- containing amino acids
have a high specific activity (500-1000 Ci/mmol), a relatively strong decay, a half-life of 87 days, and a fairly low cost. Methionine and cysteine
are supplied in aqueous solution containing 0.1 o/o ß-mercaptoethanol ( 12
mM). Cells can only talerate 0.8 mM ß-mercaptoethanol or 500 )lCi/ml.
One should avoid repetitive freeze-thawing of the isotope. The tritiated
amino acids are the second choice (20-200 Ci/mmol). The 14 C-amino