- MA1-087 - Invitrogen Antibodies HNRNPK antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), PANC-1 (Lane 2), MCF7 (Lane 3), U-2 OS (Lane 4), U-87 MG (Lane 5), IMR-32 (Lane 6), HT-29 (Lane 7) and tissue extract of Rat Testis (Lane 10). The blot was probed with Anti-hnRNP K Mouse Monoclonal Antibody (Product # MA1-087, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 51 kDa band corresponding to hnRNP K was observed across the cell lines and tissue tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of hnRNP-K was performed by loading 10 µg of whole cell human BJ fibroblast protein lysate and run on a 4-12% BTE gel. Proteins were transferred to PVDF membrane. Membrane was blocked with Pierce™ Protein-Free T20 (PBS) Blocking Buffer (Product # 37573). hnRNP-K was detected at approximately 65 kDa using a hnRNP-K monoclonal antibody (Product # MA1-087) at a dilution of 1:1000 followed by a 1:10,000 dilution of anti-rabbit HRP. Chemiluminescent detection was performed using SuperSignal West Pico PLUS substrate (Product # 34580). Data courtesy of Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of hnRNP K was achieved by transfecting HeLa cells with hnRNP K specific validated siRNAs (Silencer® select Product # s6738, s6739 and s6737). Western blot analysis (Fig. 1) was performed using whole cell extracts from the hnRNP K knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-hnRNP K, Monoclonal Antibody (Product # MA1-087, 1 µg/mL) and Goat Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. 2). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to hnRNP K.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of hnRNP K was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with hnRNP K (F45 P9 C7) Mouse Monoclonal Antibody (Product # MA1-087) at 5µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) 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 shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry of hnRNP-K expression in colon carcinoma using Product # MA1-087.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of hnRNPK was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of hnRNPK monoclonal antibody (Product # MA1-087) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a hnRNPK monoclonal antibody (Product # MA1-087) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

MA1-087