Human Granzyme H HEK293 Overexpression Lysate 产品信息
Product Description
This Human Granzyme H overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Granzyme H protein (Cat: 10348-H08H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the human Granzyme H (NP_219491.1) (Met 1-Leu 246) with a C-terminal polyhistidine tag was expressed.
Molecule Mass
The secreted recombinant human Granzyme H comprises 239 amino acids with a predicted molecular mass of 26.7 kDa. As a result of glycosylation, rh Granzyme H migrates as an approximately 36 kDa band in SDS-PAGE under reducing conditions.
Human Granzyme H HEK293 Overexpression Lysate Usage Guide
Preparation Method
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.
Lysis Buffer
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.
Recommend Usage
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.
Sample Buffer
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.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
Human Granzyme H HEK293 Overexpression Lysate Alternative Names
Human CCP-X Overexpression Lysate;Human CGL-2 Overexpression Lysate;Human CSP-C Overexpression Lysate;Human CTLA1 Overexpression Lysate;Human CTSGL2 Overexpression Lysate;Human GZMH Overexpression Lysate
Granzyme H Background Information
Granzymes are key components of the immune response that play important roles in eliminating host cells infected by intracellular pathogens. Several granzymes are potent inducers of cell death. A total of eight granzymes (A-G and M) have been identified in the mouse, but only five are known in humans (A, B, H, M and granzyme 3), and granzyme H appears to be specifically human. Human granzyme H is a neutral serine protease that is expressed predominantly in the lymphokine-activated killer (LAK)/natural?killer (NK) compartment of the immune system. In adenovirus-infected cells in which granzyme B (gzmB) and downstream apoptosis pathways are inhibited, granzyme H directly cleaves the adenovirus DNA-binding protein (DBP), a viral component absolutely required for viral DNA replication. This virus demonstrated that gzmH directly induces an important decay in viral DNA replication. Interestingly, gzmH also cleaves the adenovirus 1K assembly protein, a major inhibitor of gzmB, and relieves gzmB inhibition. Granzyme H has a very high amino acid identity (>9%) with many portions of the granzyme B sequence, particularly near the amino terminus of the molecule despite performing a distinct enzymic function.
References
Meier M., et al.,(1990), Cloning of a gene that encodes a new member of the human cytotoxic cell protease family. Biochemistry 29:4042-4049. Haddad P., et al., (1991), Structure and evolutionary origin of the human granzyme H gene.Int. Immunol. 3:57-66. Klein J.L., et al.,(1990), Characterization of a novel, human cytotoxic lymphocyte-specific serine protease cDNA clone (CSP-C).Tissue Antigens 35:220-228.
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