32-5200

antibody from Invitrogen Antibodies

Targeting: NPM1 B23, NPM

Provider product page for 32-5200

Western blot

ELISA

Immunoprecipitation

Antibody data

Antibody Data
Antigen structure
References [101]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [2]
Immunohistochemistry [1]
Flow cytometry [1]
Other assay [7]

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Product number: 32-5200 - Provider product page
Provider: Invitrogen Antibodies
Product name: NPM1 Monoclonal Antibody (FC-61991)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: This antibody reacts with the C-terminus of Nucleophosmin (B23). Positive controls used: HeLa, K562, Jurkat, MCF-7, and HL60 cell lysates). In western blots the antibody recognizes a band at ~37 kDa.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: FC-61991
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

NPM1 protein structure - 32-5200 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on nuclear enriched cell extracts of K-562 (Lane 1), HCT 116 (Lane 2), HeLa (Lane 3), HEK-293 (Lane 4), Neuro-2a (Lane 5), NTERA-2 cl.D1 (Lane 6), SH-SY5Y (lane 7), PC-12 (lane 8) and NIH/3T3 (Lane 9). Blots were probed with Anti- Nucleophosmin Mouse Monoclonal Antibody (Product # 32-5200, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conj µgate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A ~ 37 kDa band corresponding to Nucleophosmin was observed across all cell lines. An additional ~ 33 kDa band corresponding to Nucleophosmin isoform was observed in HCT 116 (Lane 2), HEK-293 (Lane 4), Neuro-2a (Lane 5), NTERA-2 cl.D1 (Lane 6) and SH-SY5Y (lane 7). Bands of ~210 kDa observed in K-562 (Lane 1), HeLa (Lane 3) and PC-12 (lane 8) could be due to oligomerisation of Nucleophosmin. Protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were transferred onto a nitrocellulose membrane and 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)
Main image
Experimental details: Knockdown of NPM1 was achieved by transfecting HeLa with NPM1 specific siRNAs (Silencer® select Product # s9677, s9676). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the NPM1 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with NPM1 Monoclonal Antibody (FC-61991) (Product # 32-5200, 0.5 ug/mL) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot for the 36 kDa band is shown in Fig. b and for the 230 kDa band in Fig. c. Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to NPM1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Nucleophosmin 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 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Nucleophosmin (FC-61991) Mouse Monoclonal Antibody (Product # 32-5200) at 2 µg/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 conj µgate (Product # A28175) 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nucleolar localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Nucleophosmin (green) in HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 3% Blocker BSA (Product # 37525) in PBS for 30 minutes at room temperature. Cells were stained with a Nucleophosmin monoclonal antibody (Product # 32-5200) at a concentration of 10 µg/mL for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:1000 for 1 hour at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of NPM1 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of NPM1 monoclonal antibody (Product # 325200) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D) and washed extensively. They were then eluted and analyzed using the Jess Simple Western system. Lane 1 is input, lane 2 IP without antibody and lane 3 IP with antibody. Target was detected a NPM1 monoclonal antibody (Product # 325200) at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA immunoprecipitation (RIP) western of NPM1 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of NPM1 monoclonal antibody (Product # 325200) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D) and washed extensively. They were then eluted and analyzed using the Jess Simple Western system. Lane 1 is input, lane 2 IP without antibody and lane 3 IP with antibody. Target was detected a NPM1 monoclonal antibody (Product # 325200) at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 P-STAT5 regulates p53/MDM2 functions and cell survival through NPM1 protein. ( a ) HEK 293T cells (6 x 10 5 per well) transfected with either RFP-STAT5A or RFP-backbone vectors were harvested at 24 h after transfection, and the cell lysates were subjected to immunoblotting assays for detection of expression levels of p53 and its phosphorylation levels at serine 15 and threonine 18, as well as the expression levels of MDM2 and its phosphorylation level at serine 166. ( b ) Six hours after transfection with either RFP-STAT5A or RFP-backbone vectors, HEK 293T cells (6 x 10 5 per well) were incubated overnight with inhibitor 573108 at a concentration of 340 mu M and then harvested and lysed for immunoblotting assays to examine the expression levels of p53 and MDM2 as well as their relevant phosphorylation levels. ( c and d ) HEK 293T cells (6 x 10 5 per well) were transfected with RFP-STAT5AY694F mutant vector for 24 h, harvested and lysed for immunoblotting assays on the expression levels of MDM2, p53 and p21. ( e ) HEK 293T cells (6 x 10 5 per well) were co-transfected with RFP-wtSTAT5A and FLAG-NPM1 vectors for 24 h, harvested and lysed for immunoblotting assays on the p53 expression levels. ( f ) MCF-7 cells (2 x 10 6 per well) were plated on 10 cm plate on day 0 and then lentivirally transduced with pLVX-IRES-mCherry-wtSTAT5 or pLVX-IRES-mCherry-STAT5Y694F on day 1. The cells were harvested 72 h after the transduction and lysed for immunoblotting assays. ( g ) HEK 2

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA immunoprecipitation (RIP) western of NPM1 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of NPM1 monoclonal antibody (Product # 325200) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D) and washed extensively. They were then eluted and analyzed using the Jess Simple Western system. Lane 1 is input, lane 2 IP without antibody and lane 3 IP with antibody. Target was detected a NPM1 monoclonal antibody (Product # 325200) at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).