Nestin antibody

• Catalog Number: orb345408
• Category: Antibodies
• Description: Nestin antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, IHC, WB
• Reactivity: Human, Mouse
• Isotype: IgG
• Immunogen: Anti-Nestin affinity purified antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to amino acids 1484-1500 of human Nestin protein.
• Concentration: 1.23 mg/mL
• Dilution range: ELISA: 1:20,000 - 1:80,000, IHC: 5ug/ml, WB: 1:500 - 1:3,000
• Form/Appearance: Liquid (sterile filtered)
• Purity: This affinity purified antibody is directed against human Nestin protein. The product was affinity purified from monospecific antiserum by immunoaffinity purification. A BLAST analysis was used to suggest cross reactivity with Nestin protein from human and Mesocricetus auratus (100% homology). Based on protein sequence homology also expect partial reactivity against Nestin homologues from mouse (93%) and rat (88%). Reactivity against homologues from other sources is not known.
• Conjugation: Unconjugated
• UniProt ID: P48681
• NCBI: 38176300
• Storage: Store anti-Nestin at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
• Buffer/Preservatives: 0.01% (w/v) Sodium Azide
• Alternative names: rabbit anti-Nestin antibody, rabbit anti-NES antib
• Note: For research use only
• Application notes: Affinity purified Anti-Nestin has been tested for use in ELISA, Immunohistochemistry (IHC), and western blot. Specific conditions for reactivity should be optimized by the end user. Expect a band approximately 200-220 kDa in size corresponding to Nestin protein by western blotting in the appropriate cell lysate or extract.
• Expiration Date: 12 months from date of receipt.

ELISA results of purified Rabbit anti-Nestin Antibody tested against BSA-conjugated peptide of immunizing peptide. Each well was coated in duplicate with 0.1 µg of conjugate. The starting dilution of antibody was 5 µg/ml and the X-axis represents the Log10 of a 3-fold dilution. This titration is a 4-parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using 3% fish gel, Goat anti-Rabbit IgG Antibody Peroxidase Conjugated (Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins) (p/n orb347654) and TMB ELISA Peroxidase Substrate (p/n orb348651).

Immunohistochemistry with Anti-nestin antibody at 40X (left) and 20X (right) Tissue: Brain and Cerebellum (right) Fixation: FFPE buffered formalin 10% conc Antigen retrieval: Heat, Citrate pH 6.2. Pressure Cooker Primary antibody: 20 ug/ml 1 hour @ room T Secondary antibody: Goat anti Rabbit Polymer HRP Prediluted by the manufacturer 30 min. @ room T Staining: antibody as precipitated red signal with a hematoxylin purple nuclear counterstain.

Immunohistochemistry with anti-nestin antibody showing nestin staining in cytoplasm of ductal epithelium of human salivary gland (B) and in nucleus and cytoplasm of human breast tissue (C). Formalin fixed/paraffin embedded sections were subjected to heat induced epitope retrieval (HIER) at pH 6.2 and then incubated with rabbit anti-nestin antibody at 4.0 µg/ml for 60 minutes. The reaction was developed using MACH 1 universal HRP polymer detection system and visualized with 3'3-diamino-benzidine substrate (DAB).

Immunohistochemistry with Anti-Nestin antibody. Tissue: mouse kidney (Left) and human kidney (Right). Fixation: formalin-fixed, paraffin-embedded tissue. Antigen retrieval: heat-induced. Primary antibody: 5 µg/ml. Staining: antibody as precipitated red signal with a hematoxylin purple nuclear counterstain.

Western blot using Biorbyt's Affinity Purified anti-Nestin antibody shows detection of a band ~220 kDa corresponding to human Nestin (arrowhead). Undifferentiated HCN-1A human brain cortical neuron neuronal progenitor lysate (lane 1), or differentiated HCN-1A human brain cortical neuron neuronal progenitor lysate (lane 2) were separated by SDS-PAGE using 4-20% gradient gel. After transfer onto nitrocellulose, the membrane was blocked and then probed with the primary antibody diluted to 1:2000 overnight at 4°C. The membrane was then washed and reacted with a 1:10000 dilution of peroxidase conjugated affinity purified Gt-a-Rabbit IgG [H&L] MX (orb347673) for 45 min at room temperature. Image was captured using film. Other detection systems will yield similar results.

Western blot using Biorbyt's Affinity Purified anti-Nestin antibody shows detection of a band ~220 kDa corresponding to mouse Nestin (arrowhead). Approximately 30 g of MEF whole cell lysate was separated by SDS-PAGE using a 4-20% gradient gel. After transfer onto nitrocellulose, the membrane was blocked and then probed with the primary antibody diluted to 1:2000 overnight at 4°C. The membrane was then washed and reacted with a 1:10000 dilution of IRDye™800 conjugated Gt-a-Rabbit IgG [H&L] MX for 45 min at room temperature.