Certificate of Analysis & Data Sheet

Recombinant human prolactin, del 1-9 G129R mutant consists of one polypeptide chain containing 190 amino and an additional Ala at N-terminus acids and having a molecular mass of ~ 22 kDa, was modified by additional 12 mutations aimed to increase its antagonistic avtivity and purified by proprietary chromatographic techniques as described by Solomon et al. Endocrinology (in press).

E. coli

Lyophylized white powder

The protein was lyophilized from a concentrated (1-2mg/ml) solution with 0.02-0.03% NaHCO3.

It is recommended to reconstitute the lyophilized hPRL in sterile water or 0.4% NaHCO3 adjusted tp pH 8-9, not less than 100µg/ml, which can then be further diluted to other aqueous solutions, preferably in presence of carrier protein.

Lyophilized del 1-9 G129R hPRL antagonist although stable at room temperature for several weeks, should be stored desiccated below -18C. Upon reconstitution at > 0.1 hPRL mg/ml and up to 3 mg/ml and filter sterilization hPRL can be stored at 4C for several weeks.

Greater than 97.0% as determined by:
(a) Gel filtration analysis (size exclusion chromtography).
(b) Analysis by reducing and non-reducing SDS-PAGE gel.

The sequence of the first six N-terminal amino acids was determined and was found to be Ala-Arg-Ser-Gln-Val-Thr

The purified del 1-9 G129R hPRL superactive antagonists consists of > 95% monomers as determined by gel-filtration chromatography.

PLR’s del 1-9 G129R hPRL super active antagonist is fully biologically active as evidenced by inhibiting hPRL-induced proliferation of Nb2 cells or Baf3 cells stably transfected with hPRL receptors. It also interacts at 1:1 molar ratio with human prolactin receptor extracellular domain as documented by SEC and SPR (Biacore analysis). Its is ~ 100 fold more potent than 1-9 G129R hPRL in Ba/F3 cells and > 1000-fold potent in Nb2 cells.

Less than 0.1 ng/µg (IEU/µg) of del 1-9 G129R hPRL

Protein content: Protein quantitation was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.96 as the extinction coefficient for a 0.1% (1mg/ml) solution at pH 8.0. This value is calculated by theDNAman computer analysis program of protein sequences.

This material is offered by PLR for laboratory research