Nucleolin/NCL Antibody

• Catalog Number: orb570341
• Category: Antibodies
• Description: Nucleolin/NCL Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, FC, ICC, IF, IHC, WB
• Reactivity: Human, Monkey, Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: E.coli-derived human Nucleolin/NCL recombinant protein (Position: K219-A629).
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.25-0.5μg/ml Immunohistochemistry (Paraffin-embedded Section), 1-2μg/ml Immunocytochemistry/Immunofluorescence, 2μg/ml, Human Flow Cytometry, 1-3μg/1x106 cells Direct ELISA, 0.1-0.5μg/ml
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 100-110 kDa
• UniProt ID: P19338
• Storage: Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.
• Alternative names: Nucleolin; Protein C23; NCL
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. By Heat: Boiling the paraffin sections in 10mM citrate buffer, pH6.0, for 20mins is required for the staining of formalin/paraffin sections. Other applications have not been tested. Optimal dilutions should be determined by end users. Add 0.2ml of distilled water will yield a concentration of 500ug/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of NCL using anti-NCL antibody (orb570341). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel)/90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human K562 whole cell lysates, Lane 2: human HeLa whole cell lysates, Lane 3: monkey COS-7 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NCL antigen affinity purified polyclonal antibody (Catalog # orb570341) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # orb90503) with Tanon 5200 system. A specific band was detected for NCL at approximately 100-110 kDa. The expected band size for NCL is at 63 kDa.

IHC analysis of NCL using anti-NCL antibody (orb570341). NCL was detected in a paraffin-embedded section of human metaplasia of squamous cells of the renal pelvis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-NCL Antibody (orb570341) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IHC analysis of NCL using anti-NCL antibody (orb570341). NCL was detected in a paraffin-embedded section of mouse pancreas tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-NCL Antibody (orb570341) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IHC analysis of NCL using anti-NCL antibody (orb570341). NCL was detected in a paraffin-embedded section of rat pancreas tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-NCL Antibody (orb570341) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

IF analysis of NCL using anti-NCL antibody (orb570341) and anti-Tubulin beta antibody (orb610920). NCL was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (orb90553) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-NCL Antibody (orb570341) and mouse anti-Tubulin beta Antibody (orb610920) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG and DyLight?594 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of HL-60 cells using anti-NCL antibody (orb570341). Overlay histogram showing HL-60 cells stained with orb570341 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-NCL Antibody (orb570341, 1 μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10 μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.