PA5-92992

antibody from Invitrogen Antibodies

Targeting: GNL3 C77032, E2IG3, MGC800, NS

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Data presented on provider website)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-92992

Provider

Invitrogen Antibodies

Product name

Nucleostemin Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Recombinant full-length protein

Description

Immunogen sequence: MTCHKRYKIQ KKVREHHRKL RKEAKKRGHK KPRKDPGVPN SAPFKEALLR EAELRKQRLE ELKQQQKLDR QKELEKKRKL ETNPDIKPSN VEPMEKEFGL CKTENKAKSG KQNSKKLYCQ ELKKVIEASD VVLEVLDARD PLGCRCPQVE EAIVQSGQKK LVLILNKSDL VPKENLESWL NYLKKELPTV VFRASTKPKD KGKITKRVKA KKNAAPFRSE VCFGKEGLWK LLGGFQETCS; Positive Samples: 293T; Cellular Location: Nucleus, nucleolus

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

0.2 mg/mL

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of extracts of 293T cells, using GNL3 Polyclonal antibody (Product # PA5-92992) at 1:1000 dilution. Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) at 1:10000 dilution. Lysates/proteins: 25ug per lane. Blocking buffer: 3% nonfat dry milk in TBST. Exposure time: 90s.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis of Nucleostemin in extracts of 293T cells using Nucleostemin Polyclonal Antibody (Product # PA5-92992) at a dilution of 1:1000. A HRP Goat Anti-Rabbit IgG (H+L) secondary antibody was used at a dilution of 1:10,000. Lysates/proteins: 25 µg per lane. Blocking buffer: 3% nonfat dry milk in TBST.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of Guanine nucleotide-binding protein-like 3 was achieved by transfecting U-2 OS with Guanine nucleotide-binding protein-like 3 specific siRNAs (Silencer® select Product # s25421, s25422). Western blot analysis (Fig. a) was performed using nuclear enriched extracts from the Guanine nucleotide-binding protein-like 3 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Nucleostemin Polyclonal Antibody (Product # PA5-92992, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). The decrease in signal upon siRNA mediated knock down confirms that the antibody is specific to Guanine nucleotide-binding protein-like 3.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Nucleostemin (GNL3) Polyclonal Antibody (Product # PA5-92992) and a 62 kDa band corresponding to Guanine nucleotide-binding protein-like 3 was observed across all the cell lines tested. Nuclear enriched extracts (30 µg lysate) of U-2 OS (Lane 1), H1299 (Lane 2), NCCIT (Lane 3), NTERA-2 cl.D1 (Lane 4), Raji (Lane 5), HCT 116 (Lane 6), SK-O-V3 (Lane 7), BeWo (Lane 8), HEK-293 (Lane 9) 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 # A27036,1:20,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of Nucleostemin was performed in MCF7 cells using Nucleostemin Polyclonal Antibody (Product # PA5-92992).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Guanine nucleotide-binding protein-like 3 was performed using 70% confluent log phase U-2 OS 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 Nucleostemin Polyclonal Antibody (Product # PA5-92992) at 1:100 dilution 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 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing nucleolar as well as nucleoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al. / Methods 115 (2017) 28-41).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation analysis of Nucleostemin was performed in 200 µg extracts of 293T cells using Nucleostemin Polyclonal Antibody (Product # PA5-92992). Western blot was performed from the immunoprecipitate using Nucleostemin Polyclonal Antibody at a dilution of 1:500.