Detection of Bovine SPAM1 by Western Blot. Western blot shows lysates of recombinant bovine SPAM1 (Catalog # 6436-GH). PVDF membrane was probed with 1 µg/mL of Sheep Anti-Bovine SPAM1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6436) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for SPAM1 at approximately 78 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.
Preparation and Storage
Sterile PBS to a final concentration of 0.2 mg/mL.
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
Stability & Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.
Sperm adhesion molecule 1 (SPAM1), also known as PH-20 hyaluronidase (1), is encoded by one of the six hyaluronidase-like genes (1, 2, 3). SPAM1 is a GPI‑anchored enzyme located on the sperm surface and inner acrosomal membrane (4). SPAM1 degrades hyaluronic acid (HA), a major structural glycosaminoglycan found in extracellular matrices and basement membranes. The enzyme activity enables sperm to penetrate through the HA-rich cumulus cell layer surrounding the oocyte and therefore facilitates the fertilization process (5). However, detailed enzymatic analysis of this enzyme is hindered by the limited techniques/methods available to monitor the HA degradation products (6). A novel method for analyzing SPAM1 activity was utilized here. Because of the structural similarity between HA (repeating units of GlcA beta 1-3GlcNAc) and chondroitin sulfate (repeating units of GlcA beta 1-3GalNAc), the enzyme is also able to hydrolyze chondroitin sulfate. In this assay, radiolabeled chondroitin sulfate was digested with recombinant SPAM1. Degradation products were then separated using a polyacrylamide electrophoresis and visualized with an X-ray film (7). The bovine SPAM1 is 63% identical to human homologue in sequence.
Lathrop, W.F. et al. (1990) J. Cell Biol. 111:2939.
Lin, Y. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:10071
Csoka, A.B. et al. (2001) Matrix Biol. 20:499.
Phelps, B.M. et al (1988) Science 240:4860
Gmachl, M. et al. (1993) FEBS Lett. 336:545.
Hofinger, E.S et al. (2007) Glycobiology 17:963.
Wu, Z.L. et al. (2010) BMC Biotechnol. 10:11. 8.
Robbins, P.W. (1962) Methods in Enzymology, Vol. V, Academic Press, Inc., New York, 964.
MacRae, I.J. et al. (2000) Biochemistry. 39:1613. 10.
The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.