PA5-17194

antibody from Invitrogen Antibodies

Targeting: CENPA CenH3, CENP-A

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-17194

Provider

Invitrogen Antibodies

Product name

CENPA Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

It is not recommended to aliquot this antibody.

Concentration

238 µg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of CENPA was achieved by transfecting HT-29 with CENPA specific siRNAs (Silencer® select Product # S2907, S2906). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the CENPA knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with CENPA Polyclonal Antibody (Product # PA5-17194, 1:1000) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). 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 CENPA.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-CENPA Polyclonal Antibody(Product # PA5-17194) and a 17kDa band corresponding to CENPA was observed across the cell lines tested. Nuclear enriched extracts (30 µg lysate) of HCT 116 (Lane 1), SW480 (Lane 2), HT-29 (Lane 3), HeLa (Lane 4), Jurkat (Lane 5), IMR32 (Lane 6) and A-431 (Lane 7) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). 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 (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of CENPA was performed using 70% confluent log phase HT-29 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with CENPA Polyclonal Antibody (Product # PA5-17194) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Centromere localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.