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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [4]
- Immunocytochemistry [2]
- Immunohistochemistry [1]
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- Product number
- PA5-83350 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Nucleostemin Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant full-length protein
- Description
- Immunogen sequence: SILFQSSGLT NGIIEEKDIH EELPKRKERK QEEREDDKDS DQETVDEEVD ENSSGMFAAE ETGEALSEET TAGEQSTRSF I
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 0.7 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of Nucleostemin by a Nucleostemin polyclonal antibody (Product # PA5-83350). Lane 1: Marker [kDa] 230, 130, 95, 72, 56, 36, 28, 17, 11 Lane 2: Human cell line RT-4 Lane 3: Human cell line U-251 MG.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of Nucleostemin by a Nucleostemin polyclonal antibody (Product # PA5-83350). Lane 1: Marker [kDa] 230, 130, 95, 72, 56, 36, 28, 17, 11 Lane 2: Human cell line RT-4 Lane 3: Human cell line U-251 MG.
- 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 # PA5-83350, 0.4 µg/mL) 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.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-Nucleostemin Polyclonal Antibody (Product # PA5-83350) 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), PC-3 (Lane 9), HEK-293 (Lane 10), U-87 MG (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 (0.4 µg/mL) 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).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of Nucleostemin in U-2 OS cells using a Nucleostemin polyclonal antibody (Product # PA5-83350). The analysis shows localization to nucleoli & nuclear bodies.
- 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 # PA5-83350) 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).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemical analysis of Nucleostemin in human oral mucosa using a Nucleostemin polyclonal antibody (Product # PA5-83350). The analysis shows strong nuclear/ nucleolar positivity in squamous epithelial cells.
