Beta-galactosidase activity measurement in HeLA cells using CPRG Beta-Galactosidase Kit

Beta-galactosidase activity was measured in HeLa-P4C5 cell lysate with the CPRG Beta-Galactosidase Kit from OZ Biosciences.

This paper demonstrates the ability of the CPRG kit from OZ Biosciences to efficienctly measure the LacZ activity in cell lines.

article reference: Clin Infect Dis. 2013 Sep;57(5):745-55.  

A Single Amino-Acid Change in a Highly Conserved Motif of gp41 Elicits HIV-1 Neutralization and Protects Against CD4 Depletion.
Petitdemange C, Achour A, Dispinseri S, Malet I, Sennepin A, Ho Tsong Fang R, Crouzet J, Marcelin AG, Calvez V, Scarlatti G, Debré P, Vieillard V.

Abstract

Background The induction of neutralizing antibodies against conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope protein is a major goal of vaccine strategies. We previously identified 3S, a critical conserved motif of gp41 that induces the NKp44L ligand of an activating NK receptor. In vivo, anti-3S antibodies protect against the natural killer (NK) cell-mediated CD4 depletion that occurs without efficient viral neutralization. Methods Specific substitutions within the 3S peptide motif were prepared by directed mutagenesis. Virus production was monitored by measuring the p24 production. Neutralization assays were performed with immune-purified antibodies from immunized mice and a cohort of HIV-infected patients. Expression of NKp44L on CD4(+) T cells and degranulation assay on activating NK cells were both performed by flow cytometry. Results Here, we show that specific substitutions in the 3S motif reduce viral infection without affecting gp41 production, while decreasing both its capacity to induce NKp44L expression on CD4(+) T cells and its sensitivity to autologous NK cells. Generation of antibodies in mice against the W614 specific position in the 3S motif elicited a capacity to neutralize cross-clade viruses, notable in its magnitude, breadth, and durability. Antibodies against this 3S variant were also detected in sera from some HIV-1-infected patients, demonstrating both neutralization activity and protection against CD4 depletion. Conclusions These findings suggest that a specific substitution in a 3S-based immunogen might allow the generation of specific antibodies, providing a foundation for a rational vaccine that combine a capacity to neutralize HIV-1 and to protect CD4(+) T cells.

CPRG ß-galactosidase assay kit:This assay kit is ideal for quantitatively measure low expression level of ß-gal. The levels of active ß-gal expression can be quickly measured by its catalytic hydrolysis of CPRG (Chlorophenol red-ß-D-galactopyranoside), substrate to a dark red product. The high sensitivity of this substrate improves the measurement of ß-gal activity when the reporter gene expression is low. (Absorbance at 570-595 nm)