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Abstract
A new preparative scale procedure is described for the efficient purification of Thyroid stimulating hormone (TSH) from buffalo pituitary glands. The methodology involves the use of hydrophobic interaction chromatography for the separation of TSH from LH which is more abundant than TSH in the pituitary gland. The two hormones were isolated from the pituitary extract by ammonium sulphate precipitation and were then separated on a Phenyl-Sepharose column. The fractions obtained from Phenyl-Sepharose chromatography were assessed using a Uno-Q column on FPLC where enrichment of the TSH in the PS-2 fraction was evident. The final removal of the trace amounts of the LH was done by ion exchange chromatography in tandem on CM-Sephadex and DEAE-Sephacel columns. All the chromatography fractions were assessed in a heterologous direct binding ELISA using bTSH and buLH as the reference hormones and anti ovine TSH beta and anti bovine LH beta as the antisera. The purified fraction (DEAE-0.5 M peak) when checked exhibited no LH immuno reactivity in a western blot against anti bovine LH beta antiserum. Forty two milligrams of a 200 fold purified buTSH per kg wet glands was obtained.