- MA3-053 - Invitrogen Antibodies CBX1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HP1-beta was performed by loading 0.5 µg of recombinant HP1-gamma (lane 1), HP1-beta (lane 2), and HP1-alpha (lane 3) in reducing sample buffer (Product # 39000) and Page Ruler Plus Protein Ladder (Product # 26619) onto a Novex 4-20% Tris-Glycine polyacrylamide gel (Product # WT4201BX10). Proteins were transferred to PVDF membrane (Product # 88518) with Transfer Buffer (Product # 84731) using the G2 Fast Blotter (Product # 62288). Membrane was blocked in StartingBlock T20 (Product # 37543) for one hour at room temperature. HP1-beta was detected at approximately 25 kDa using HP1-beta monoclonal antibody (Product # MA3-053) at a dilution of 1:2000 overnight at 4°C on a rocking platform, followed by a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:5,000 for one hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076) and the myECL Imager (Product # 62236).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HP1 was achieved by transfecting MCF7 with HP1 specific siRNAs (Silencer® select Product # s21549). Western blot analysis (Fig. a) was performed using whole cell extracts from the HP1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HP1 Monoclonal Antibody (Product # MA3-053, 1:2000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to HP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti- HP1 Monoclonal Antibody(Product # MA3-053) and a 25 kDa band corresponding to HP1 was observed across the cell lines and tissues tested. Modified whole cell extracts (1% SDS) (30ug lysate) of HeLa (Lane 1), MCF7 (Lane 2), A-431 (Lane 3), U-2 OS (Lane 4), PC-3 (Lane 5), Raji (Lane 6), IMR32 (Lane 7), NIH/3T3 (Lane 8), Hep G2 (Lane 9), Mouse Liver (Lane 10) and Rat Liver (Lane 11) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of HP1-beta (green) in HeLa cells. The cells were fixed with formalin for 15 minutes, permeabilized with 1X Permeablilization buffer (Product # 8408400) for 15 minutes, and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were stained with HP1-beta monoclonal antibody (Product # MA3-053) at a dilution of 1:250 in blocking buffer for one hour at room temperature, washed with 1X TBS Tween 20 Buffer (Product # 28360), followed by incubation with DyLight 488 goat anti-mouse IgG secondary antibody (green, Product # A28175) at a dilution of 1:2000 and Hoechst 33342 dye (blue, Product # 62249) at a dilution of 1:5000 for 30 minutes at room temperature. Images were taken on a Thermo Scientific ToxInsight at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin immunoprecipitation analysis of HIV1 HP1-beta was performed using cross-linked chromatin from Human 5A8 J-lat T lymphocytes culture latently infected with HIV1 and treated with 10 µg/mL PHA (phytohemaglutin) for 0, 4, and 8 hours. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of HP1-beta monoclonal antibody (Product # MA3-053). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers shown to amplify exon-1 of the Egr1 gene or HIV gag gene. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. Schematic representations of the Egr1 and HIV1 gag locus are shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by the primers are represented by black bars. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: ELISA analysis of HP1-beta was performed by coating wells of a 96-well plate with 100 µL per well of recombinant HP1-beta protein or BSA (Product # 23209) diluted in carbonate/bicarbonate buffer (Product # 28382), at a concentration of 1 µg/mL. Wells of the plate were washed, blocked with Superblock blocking buffer (Product # 37536), and incubated with 100 µL per well of a mouse anti-HP1-beta monoclonal antibody (Product # MA3-053) starting at a concentration of 1 µg/mL and serially diluting 2-fold to a concentration of 1 ng/mL for 1 hour at 37°C. The plate was washed, then incubated with 100 µL per well of goat anti-mouse IgG SuperclonalTM secondary antibody (Product # A28174) at a dilution of 1:10,000 for 1 hour at 37°C followed by washing. Detection was performed using 1-Step Ultra TMB substrate (Product # 34028) for 5 minutes at room temperature in the dark. The reaction was stopped with 0.16M sulfuric acid, and absorbances were read on a spectrophotometer at 450-550 nm.

MA3-053

Antibody data