PA3-16875

antibody from Invitrogen Antibodies

Targeting: NCL C23, Nsr1

Immunoelectron microscopy

Chromatin Immunoprecipitation

Antibody data

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Validation data

Product number: PA3-16875 - Provider product page
Provider: Invitrogen Antibodies
Product name: Nucleolin Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Other
Description: Heat mediated antigen retrieval is recommended for IHC on paraffin embedded tissues. Suggested positive control: PD31 murine pre-B cells for immunofluorescence and crude PD31 nuclear extracts for Western Blot.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

NCL protein structure - PA3-16875 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot detection of nucleolin in crude PD31 nuclear extracts.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Nucleolin was achieved by transfecting HeLa cells with Nucleolin specific siRNAs (Silencer® select Product # s9313). Western blot analysis (Fig. a) was performed using Nuclear extracts from the Nucleolin knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Nucleolin Polyclonal Antibody (Product # PA3-16875, 1:4000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/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 Nucleolin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on modified whole cell extracts (30 µg lysate) of HeLa (Lane 1), U-2 OS (Lane 2), U-87 MG (Lane 3), T98G (Lane 4), SH-SY5Y (Lane 5) and NTERA-2 (Lane 6). The blot was probed with Anti-Nucleolin Polyclonal Antibody (Product # PA3-16875, 1:4000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 100 kDa band corresponding to Nucleolin was observed across the cell lines tested. An additional band around 75 kDa was observed in the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Nucleolin in crude PD31 nuclear extracts. Sample was incubated in Nucleolin polyclonal antibody (Product # PA3-16875).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Nucleolin was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR940415_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Nucleolin was performed by loading 60 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Nucleolin KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Nucleolin Polyclonal Antibody (Product # PA3-16875, 1:8000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 (Product # A44115). Chemiluminescent detection was performed usingSuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Nucleolin. Analysis confirmed that the band observed in lane 3 below ~100 kDa is the truncated protein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Nucleolin in 0.5 mg/mL HeLa lysate. Samples were incubated in Nucleolin polyclonal antibody (Product # PA3-16875). This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: In-situ immunofluorescent staining of PD31 murine pre-B cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of Nucleolin in PD31 murine pre-B cells. Samples were incubated in Nucleolin polyclonal antibody (Product # PA3-16875).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of Nucleolin in PD31 murine pre-B cells. Samples were incubated in Nucleolin polyclonal antibody (Product # PA3-16875).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Nucleolin was performed using 70% confluent log phase U-2 OS cells treated with Heat Shock (44 degrees for 3 hrs). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Nucleolin Polyclonal Antibody (Product # PA3-16875) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing translocation of Nucleolin in to the nucleus upon Heat Shock. Panel e represents the control cells showing exclusive nucleolar staining. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.