PA5-40129

antibody from Invitrogen Antibodies

Targeting: SHARPIN DKFZP434N1923, SIPL1

Antibody data

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Validation data

Product number: PA5-40129 - Provider product page
Provider: Invitrogen Antibodies
Product name: SHARPIN Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Recombinant full-length protein
Description: Recommended positive controls: K562 and THP-1 cells. Predicted reactivity: Chimpanzee (96%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.
Reactivity: Human
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: 3.41 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

SHARPIN protein structure - PA5-40129 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: SHARPIN Polyclonal Antibody detects SHARPIN protein by western blot analysis. Various whole cell extracts (30 µg) were separated by 10% SDS-PAGE, and the membrane was blotted with SHARPIN Polyclonal Antibody (Product # PA5-40129) diluted at a dilution of 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-SHARPIN Polyclonal Antibody (Product # PA5-40129) and a 42 kDa band corresponding to SHARPIN was observed across the cell lines tested. An uncharacterized band of ~ 35 kDa was also observed in all the cell lines. Whole cell extracts (30 µg lysate) of PC-3 (Lane1), DU 145 (Lane 2), MDA-MB-231 (Lane 3), MCF7 (Lane 4), HeLa (Lane 5), A549 (Lane 6), Jurkat (Lane 7) and THP-1 (Lane 8) were electrophoresed using Novex® NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 (Heavy Chain), 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: Knockdown of SHARPIN was achieved by transfecting A549 cells with SHARPIN specific siRNAs (Silencer® select Product # s195427, s37848). Western blot analysis (Fig. a) was performed using whole cell extracts from the SHARPIN knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-SHARPIN Polyclonal Antibody (Product # PA5-40129, 1:1000 dilution) and Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25 µg/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 SHARPIN. An uncharacterized band around 35 kDa was also observed.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of SHARPIN was performed using 70% confluent log phase A549 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 SHARPIN Polyclonal Antibody (Product # PA5-40129) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of SHARPIN was performed using 70% confluent log phase A549 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 SHARPIN Polyclonal Antibody (Product # PA5-40129) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.