Nucleostemin/GNL3 Antibody

• Catalog Number: orb865524
• Category: Antibodies
• Description: Nucleostemin/GNL3 Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, FC, ICC, IF, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: E.coli-derived human Nucleostemin/GNL3 recombinant protein (Position: R45-V549).
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.25-0.5 μg/ml, Human, Mouse, Rat Immunohistochemistry(Paraffin-embedded Section), 2-5 μg/ml, Human Immunocytochemistry/Immunofluorescence, 5 μg/ml, Human Flow Cytometry, 1-3 μg/1x106 cells, Human Direct ELISA, 0.1-0.5 μg/ml, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 65 kDa
• UniProt ID: Q9BVP2
• Storage: 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 freezing and thawing.
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. Other applications have not been tested. Optimal dilutions should be determined by end users. Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of Nucleostemin/GNL3 using anti-Nucleostemin/GNL3 antibody (orb865524). 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 HeLa whole cell lysates, Lane 2: human K562 whole cell lysates, Lane 3: human PC-3 whole cell lysates, Lane 4: human Jurkat whole cell lysates, Lane 5: rat C6 whole cell lysates, Lane 6: mouse NIH/3T3 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-Nucleostemin/GNL3 antigen affinity purified polyclonal antibody (Catalog # orb865524) 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 Nucleostemin/GNL3 at approximately 65 kDa. The expected band size for Nucleostemin/GNL3 is at 65 kDa.

IHC analysis of Nucleostemin/GNL3 using anti-Nucleostemin/GNL3 antibody (orb865524). Nucleostemin/GNL3 was detected in a paraffin-embedded section of human appendiceal adenocarcinoma 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-Nucleostemin/GNL3 Antibody (orb865524) 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 Nucleostemin/GNL3 using anti-Nucleostemin/GNL3 antibody (orb865524). Nucleostemin/GNL3 was detected in a paraffin-embedded section of human breast cancer 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-Nucleostemin/GNL3 Antibody (orb865524) 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 Nucleostemin/GNL3 using anti-Nucleostemin/GNL3 antibody (orb865524). Nucleostemin/GNL3 was detected in a paraffin-embedded section of human colonic adenocarcinoma 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-Nucleostemin/GNL3 Antibody (orb865524) 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 Nucleostemin/GNL3 using anti-Nucleostemin/GNL3 antibody (orb865524). Nucleostemin/GNL3 was detected in a paraffin-embedded section of human gall bladder adenosquamous carcinoma 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-Nucleostemin/GNL3 Antibody (orb865524) 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 Nucleostemin/GNL3 using anti-Nucleostemin/GNL3 antibody (orb865524). Nucleostemin/GNL3 was detected in an immunocytochemical section of HEP3B 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 5 μg/mL rabbit anti-Nucleostemin/GNL3 Antibody (orb865524) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The tissue section was developed using Phalloidin-iFluor 555 Conjugated. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of HeLa cells using anti-Nucleostemin/GNL3 antibody (orb865524). Overlay histogram showing HeLa cells stained with orb865524 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Nucleostemin/GNL3 Antibody (orb865524, 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.