This Human FSH Beta overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of FSH Beta protein (Cat: 11935-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the human FSHB (NP_000501.1) (Met1-Glu129) was expressed with a polyhistidine tag at the C-terminus.
The recombinant human FSHB consists 122 amino acids and predicts a molecular mass of 13.9 kDa.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube.
2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. FSHB represents a conservative vertebrate gene with a unique function and it is located in a structurally stable gene-poor region. Polymorphisms in the follicle stimulating hormone beta subunit (FSHB) and follicle stimulating hormone receptor (FSHR) genes might disturb normal spermatogenesis and affect male reproductive ability. The FSHB -211G>T genotype is a key determinant in the regulation of gonadotropins in different reproductive-endocrine pathopyhsiologies.