BD PharmingenTM Purified Mouse Anti-Human VHL

Description

Western blot analysis of VHL expression and RNAi validation of anti-VHL antibody specificity - Panel 1. Lysate prepared from HeLa S3 cells Panel 1a was blotted using Purified Mouse Anti-VHL antibody Cat. No. 564183 at concentrations of 1 lane 1 , 0.5 lane 2 , or 0.25 lane 3 mug/ml. VHL was identified as a band of 24 kDa Panel 1a . Lysates from untreated, VHL RNAi- or ERK2 RNAi-treated HeLa S3 cells Panel 1b were blotted with Purified Mouse Anti-VHL or Anti-ERK-2 Cat. No. 554095 antibody. The expressed level of VHL protein blotted with Anti-VHL antibody 2 mug/ml is downregulated in lysate prepared from HeLa S3 cells treated with VHL RNAi but not ERK2 RNAi. Similarly, ERK2 protein expression was reduced in lysate from HeLa S3 cells treated with ERK2 RNAi but not VHL RNAi when blotted with Anti-ERK2 antibody 0.5 mug/ml . Immunohistochemical staining of VHL expressed in tissue from a human renal tumor - Panel 2. Following antigen retrieval with BD Retrievagen A buffer Cat. no. 550524 , the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b, kappa Isotype Control Cat. No. 557351; Top Tissue or Purified Mouse Anti-VHL antibody Bottom Tissue using the BD Biosciences Protocol for Immunohistochemical Staining. A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin Cat. No. 550337 , Streptavidin-Horseradish Peroxidase HRP Cat. No.550946 , and the DAB Substrate Kit Cat. No. 550880 was used to develop the primary staining reagents. The tissue was counterstained with Hematoxylin. Original magnification: 20X. Flow cytometric analysis of VHL expression in VHL-transfected 293F cells - Panel 3. Untransfected dashed line histogram and human VHL-transfected solid line histogram 293F cells were fixed with BD Cytofix Fixation Buffer Cat. No. 554655 and permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 . The cells were then washed and stained with Purified Mouse Anti-Human VHL antibody followed by APC Goat Anti-Mouse Ig Cat. No. 550826 using BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD FACSCanto II Flow Cytometer System. Western blot analysis of VHL expression and RNAi validation of anti-VHL antibody specificity - Panel 1. Lysate prepared from HeLa S3 cells Panel 1a was blotted using Purified Mouse Anti-VHL antibody Cat. No. 564183 at concentrations of 1 lane 1 , 0.5 lane 2 , or 0.25 lane 3 mug/ml. VHL was identified as a band of 24 kDa Panel 1a . Lysates from untreated, VHL RNAi- or ERK2 RNAi-treated HeLa S3 cells Panel 1b were blotted with Purified Mouse Anti-VHL or Anti-ERK-2 Cat. No. 554095 antibody. The expressed level of VHL protein blotted with Anti-VHL antibody 2 mug/ml is downregulated in lysate prepared from HeLa S3 cells treated with VHL RNAi but not ERK2 RNAi. Similarly, ERK2 protein expression was reduced in lysate from HeLa S3 cells treated with ERK2 RNAi but not VHL RNAi when blotted with Anti-ERK2 antibody 0.5 mug/ml . Show More Immunohistochemical staining of VHL expressed in tissue from a human renal tumor - Panel 2. Following antigen retrieval with BD Retrievagen A buffer Cat. no. 550524 , the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b, kappa Isotype Control Cat. No. 557351; Top Tissue or Purified Mouse Anti-VHL antibody Bottom Tissue using the BD Biosciences Protocol for Immunohistochemical Staining. A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin Cat. No. 550337 , Streptavidin-Horseradish Peroxidase HRP Cat. No.550946 , and the DAB Substrate Kit Cat. No. 550880 was used to develop the primary staining reagents. The tissue was counterstained with Hematoxylin. Original magnification: 20X. Show More Flow cytometric analysis of VHL expression in VHL-transfected 293F cells - Panel 3. Untransfected dashed line histogram and human VHL-transfected solid line histogram 293F cells were fixed with BD Cytofix Fixation Buffer Cat. No. 554655 and permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 . The cells were then washed and stained with Purified Mouse Anti-Human VHL antibody followed by APC Goat Anti-Mouse Ig Cat. No. 550826 using BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD FACSCanto II Flow Cytometer System. Show More Western blot analysis of VHL expression and RNAi validation of anti-VHL antibody specificity - Panel 1. Lysate prepared from HeLa S3 cells Panel 1a was blotted using Purified Mouse Anti-VHL antibody Cat. No. 564183 at concentrations of 1 lane 1 , 0.5 lane 2 , or 0.25 lane 3 mug/ml. VHL was identified as a band of 24 kDa Panel 1a . Lysates from untreated, VHL RNAi- or ERK2 RNAi-treated HeLa S3 cells Panel 1b were blotted with Purified Mouse Anti-VHL or Anti-ERK-2 Cat. No. 554095 antibody. The expressed level of VHL protein blotted with Anti-VHL antibody 2 mug/ml is downregulated in lysate prepared from HeLa S3 cells treated with VHL RNAi but not ERK2 RNAi. Similarly, ERK2 protein expression was reduced in lysate from HeLa S3 cells treated with ERK2 RNAi but not VHL RNAi when blotted with Anti-ERK2 antibody 0.5 mug/ml . Immunohistochemical staining of VHL expressed in tissue from a human renal tumor - Panel 2. Following antigen retrieval with BD Retrievagen A buffer Cat. no. 550524 , the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b, kappa Isotype Control Cat. No. 557351; Top Tissue or Purified Mouse Anti-VHL antibody Bottom Tissue using the BD Biosciences Protocol for Immunohistochemical Staining. A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin Cat. No. 550337 , Streptavidin-Horseradish Peroxidase HRP Cat. No.550946 , and the DAB Substrate Kit Cat. No. 550880 was used to develop the primary staining reagents. The tissue was counterstained with Hematoxylin. Original magnification: 20X. Flow cytometric analysis of VHL expression in VHL-transfected 293F cells - Panel 3. Untransfected dashed line histogram and human VHL-transfected solid line histogram 293F cells were fixed with BD Cytofix Fixation Buffer Cat. No. 554655 and permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 . The cells were then washed and stained with Purified Mouse Anti-Human VHL antibody followed by APC Goat Anti-Mouse Ig Cat. No. 550826 using BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD FACSCanto II Flow Cytometer System.