PA5-21415

antibody from Invitrogen Antibodies

Targeting: NXF1 DKFZp667O0311, Mex67, TAP

Provider product page for PA5-21415

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [1]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [4]
Immunohistochemistry [1]
Other assay [2]

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Product number: PA5-21415 - Provider product page
Provider: Invitrogen Antibodies
Product name: TAP Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Recombinant protein fragment
Description: Recommended positive controls: HeLa, HepG2, Molt-4.
Concentration: 0.3 mg/mL

NXF1 protein structure - PA5-21415 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using TAP Polyclonal Antibody (Product # PA5-21415). Sample (30 µg of whole cell lysate). Lane A: Hela. Lane B: Molt-4 . 7.5% SDS PAGE. TAP Polyclonal Antibody (Product # PA5-21415) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of TAP was achieved by transfecting HeLa cells with TAP specific siRNAs (Silencer® select Product # s35502, s20533). Western blot analysis (Fig. a) was performed using whole cell extracts from the TAP knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2), and untransfected cells (lane 1). The blot was probed with TAP Polyclonal Antibody (Product # PA5-2145, 1:1000) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in the histogram (Fig. b). A decrease in signal upon siRNA mediated knock down confirms that antibody is specific to TAP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-TAP Polyclonal Antibody (Product # PA5-21415) and a 70 kDa band corresponding to TAP was observed across all cell lines tested. whole cell extracts (40 µg lysate) of HeLa (Lane 1), MCF-7 (Lane 2), A-431 (Lane 3), MOLT-4 (Lane 4), SH-SY5Y (Lane 5), SK-O-V3 (Lane 6), NIH/3T3 (Lane 7), Mouse Placenta (Lane 8), Mouse Heart (Lane 9), Mouse Kidney (Lane 10) and Rat Heart (Lane 11) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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)
Main image
Experimental details: Immunofluorescent analysis of TAP in paraformaldehyde-fixed HeLa cells using a TAP polyclonal antibody (Product # PA5-21415) at a 1:200 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of TAP was achieved by transfecting HeLa cells with TAP specific siRNA (Silencer® select Product # S35502, S20533). Immunofluorescence analysis was performed on untransfected HeLa cells (panels a,d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with TAP specific siRNA (panels c,f). Cells were fixed, permeabilized, and labelled with TAP Polyclonal Antibody (Product # PA5-21415), (1:100 dilution) followed by Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790, 1:2,000 dilution). Nuclei (blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (Red) staining. Partial reduction of specific signal was observed upon siRNA mediated knockdown (panels c,f) confirming specificity of the antibody to TAP (Green). The Images were captured at 60X magnification with oil immersion.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of paraformaldehyde-fixed HeLa, using TAP (Product # PA5-21415) antibody at 1:200 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of TAP was performed using HeLa 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 1 hour at room temperature. The cells were labeled with Anti-TAP Polyclonal Antibody (Product # PA5-21415) at 1:50 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) at a dilution of 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 composite image showing Nuclear with slight cytosolic localization of TAP in HeLa cells. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification with oil immersion.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 4 CTE-gRNAs accumulate at the microtubule organizing center. (A, B). 4xCTE-gRNAs in HeLa.MS2-YFP cells cluster at the MTOC as demonstrated by co-expressing mCherry-Tubulin (A) or staining for the centrosomal marker Pericentrin (B) . Arrows highlight the marked enrichment of CTE-gRNAs to this region. (C) Single particle tracking over 10 minutes for 4xCTE-gRNAs undergoing bidirectional and rectilinear trafficking at or near the MTOC. Arrows represent individual gRNA paths over the time of imaging. Figure corresponds to S4 Movie . (D) NXF1 traffics with 4xCTE-gRNAs to the MTOC. Cells expressing RRE- or 4xCTE-gRNAs were incubated with or without 3 muM nocodazole (as indicated) for 36 h prior to fixation and detection of NXF1 using indirect immunofluorescence. Black arrows indicate regions of co-localization between NXF1 and 4xCTE-gRNAs (E) Quantification of MS2-YFP and NXF1 localization at the MTOC for the indicated conditions. For these experiments the MTOC was defined using Pericentrin immunostaining. Error bars represent the standard deviation of the mean for three (MS2-YFP, n = 50) or two (NXF1, n = 50) independent experiments. All size bars correspond to 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 5 CTE-gRNAs accumulate at centrosomes during mitosis and are subsequently partitioned to daughter cells. (A) 4xCTE-gRNAs are partitioned to daughter cells via centrosomes during cell division. A representative HeLa.MS2-YFP cell expressing 4xCTE-gRNAs and tracked through metaphase, anaphase, and telophase. Yellow arrows indicate the enriched MS2-YFP signal (green) at the spindle poles (mCherry-tubulin in red) during metaphase (Hoechst DNA stain in blue). Figure corresponds to S5 Movie . (B) Unlike 4xCTE-gRNAs, RRE-gRNAs do not accumulate at the mitotic spindle. Imaging as for (A) but for cells co-expressing RRE-gRNAs and Rev. (C,D) 4xCTE-gRNAs (MS2-YFP; green) co-localize with the centrosome marker Pericentrin (C, white) and also NXF1 (D, white) at the poles of the spindle (shown using mCherry-tubulin, red). Pericentrin and NXF1 were detected by indirect immunofluorescence. For all panels, yellow arrows highlight 4xCTE-gRNA enrichment at the centrosome. Size bars represent 10 mum.