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Removal of residual fat from the IgG fraction
Residual fat has co-precipitated during the IgG enrichment and it has to be removed before the material is ready for the affinity column. At 4C most of the fat that potentially could cause problems has clumped and floats at the surface. A simple filtering step at 4C removes this fat, plus other aggregated materials.
Conjugation of peptide to the affinity column
All our immunizing peptides have a cysteine residue. If such residue does not belong in the peptide sequence of choice, it is added to either the N-terminus or C-terminus of the peptide. We usually pick the N-terminus except when the peptide represents the N-terminus of the protein of interest.
The cysteine in the peptide provides the sulfhydryl group that will covalently bind to the iodoacetyl groups in the resin of the column. Thus the immunizing peptide gets permanently immobilized inside the column.
Blocking the remaining active sites on the column
Once all the peptide is loaded, the remaining unused iodoacetyl groups in the column are blocked by an equal volume of 50mM cysteine to the bed volume of the column for 15min at room temp. The required bed volume varies from 1ml through 4ml depending on the ELISA titres of the IgG on the immunizing peptide.
We then wash the column with 1M NaCl followed by sodium azide wash. Finally the column is washed in PBS/NaN3 before it is stored at 4C waiting to be used.
Wash the column with PBS
Pass the filtered IgG (all of it) through the column once
Wash with PBS
Wash with 0.5M NaCl in PBS
Wash with PBS
Elute with glycine-HCL pH 2.6 until collected eight 1ml fractions.
The eluted fractions remain exposed to pH2.6 for at least 30 minutes in total –an extra precaution so to comply with international export/import legislation.
Then each 1ml fractions is neutralized with 91ul 2M Tris pH8.0, 1.5M NaCl.
Protein content of the fractions are monitored by A280nm.
The column is washed with PBS and the same IgG is passed through the column again.
This routine is repeated until no more specific antibody can be recovered.
The last eluted fraction of the third round determines whether or not to continue the affinity purification process. When this fraction contains 1mg/ml protein or higher, we will continue until the next last eluted fraction drops to 0.49mg/ml protein or we finished 6 rounds, whichever came first.
All fractions containing >0.49mg/ml protein are pooled and the final yield is determined. All affinity purified antibodies are divided into aliquots with NaN3 and BSA. Antibodies with over 1mg/ml protein will not get BSA in all its aliquots so they can serve customers who need the antibody pure.