Invitrogen

PAX3 Polyclonal Antibody

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PAX3 Antibody in Immunocytochemistry (ICC/IF) — Immunofluorescent analysis of Pax3 (red) in human neural stem cells derived from PD-3 iPSCs using Gibco® PSC Neural Induction Medium (Product # A1647801). The cells were fixed and permeabilized using Image-IT® Fixation/Permeabilization kit (Product # R37602), and blocked with blocking buffer included the kit for one hour at room temperature. Cells were stained with a Pax3 polyclonal antibody (Product # PA1-107) at a dilution of 1:200 in blocking buffer for 3 hours at room temperature, and then incubated with an AlexaFluor 594- conjugated donkey anti-rabbit IgG secondary antibody (Product # A-21207) at a dilution of 1:250 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI (Product # D1306). Images were taken on an EVOS® FLoid® Cell Imaging Station at 10X magnification.

PAX3 Antibody in Immunohistochemistry (Frozen) (IHC (F)) — Immunofluorescence was performed on mouse E10.5 embryo trunk at the forelimb level. Embryos were fixed with 4%PFA in 0.1M phosphate buffer, pH 7.2 at 4C for 30 minutes, processed for cryoprotection in 20% sucrose, and embedded in OCT compound for cryosectioning at 10 µm thickness. Following sectioning, tissues were washed in PBS with 0.1% Triton-X 100 (PBT), blocked in 1% goat serum in PBT for 30 minutes at room temperature, and then probed with a Pax3 polyclonal antibody (Product # PA1-107) at a concentration of 1 µg/mL in blocking buffer overnight at 4C. Tissues were washed extensively with PBT, and detection was performed using an Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibody (red) at a dilution of 1:1000 in blocking buffer. Nuclei (blue) were stained with DAPI. Note: Staining was observed in the dorsal neural tube (symmetric two halves on the left side of the image), the dermomyotome/myotome (middle streak between symmetric halves), and limb muscle progenitors (scattered cells to the right side of the image). Data supplied courtesy of the Innovator's Program.

PAX3 Antibody in Western Blot (WB) — Western blot analysis of Pax3 was performed by loading the indicated amounts of Pax3 and Pax4 (negative control) recombinant proteins, and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. Pax3 was detected at ~53 kD using a Pax3 polyclonal antibody (Product # PA1-107) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for at least 30 minutes. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). NOTE: The presence of Pax4 recombinant protein was determined by probing a duplicate blot with a Pax4 polyclonal antibody (Product # PA1-108), and the corresponding image is on the webpage for Product # PA1-108.

PAX3 Antibody in Immunoprecipitation (IP) — Immunoprecipitation of Pax3 was performed on RIPA cell extracts originating from a 6cm dish of HeLa cells transfected for 24 hours with a Pax3-HA construct. Cells were lysed in 1 mL of RIPA buffer, and antigen-antibody complexes were formed by incubating 225 µL of HeLa Pax-HA cell extract with 2 µg of a Pax3 polyclonal antibody (Product # PA1-107) and Protein A sepharose for 4 hours at 4C. The samples were washed extensively with RIPA buffer, and eluted by boiling in SDS sample buffer. Samples were resolved on an 8% Tris-Glycine gel alongside whole cell lysate (WCL) from empty vector and Pax3-HA-transfected HeLa cells, transferred to a nitrocellulose membrane, and blocked with 2% BSA in PBST for 10 minutes at room temperature. The membrane was probed with an anti-HA polyclonal antibody overnight at 4C, washed in TBST, and probed with an HRP-conjugated anti-rabbit IgG secondary antibody. Chemiluminescent detection was performed using ECL substrate. Data supplied courtesy of the Innovator's Program.

PAX3 Antibody in Immunohistochemistry (IHC) — Fig. 1 Neuroprogenitor migration in Ntn1 , Dcc , and Robo3 knockouts. a Schematic of the migration of the dorsal spinal cord progenitors and interneurons. VZ, ventricular zone. b Cross sections of the spinal cord electroporated with Actb-gfp ( betaactin-gfp) . The closeup images are of the boxed area. The embryos were cultured for 20 h. GFP+ neurons from all three KOs migrate out of the VZ (demarcated by PAX3/7 staining) later than WT neurons. c Quantification of the ratio between GFP+ neurons within the VZ and the total GFP+ neurons. A higher percentage of neurons is seen within the VZ in all three KOs. Data are represented as the mean +- SEM (Student's t -test; **, p < 0.001; *, p < 0.05). Scale bars, 50 mum

Product Details

Applications

Tested Dilution

Publications

Western Blot (WB)

1:500-1:1,000

Immunohistochemistry (IHC)

View 2 publications 2 publications

Immunohistochemistry (Frozen) (IHC (F))

1:100-1:1,000

Immunocytochemistry (ICC/IF)

1:100-1:1,000

View 1 publication 1 publication

Immunoprecipitation (IP)

2 µg

Product Specifications

Species Reactivity

Human, Mouse

Published species

Human, Mouse

Host/Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

Amino acids 301-442 of human PAX3

Conjugate

Unconjugated

Form

Liquid

Concentration

1 mg/mL

Purification

Protein G

Storage buffer

PBS with 1mg/mL BSA, 30% glycerol

Contains

0.05% sodium azide

Storage conditions

-20°C

RRID

AB_2539881

Product Specific Information

PA1-107 has successfully been used in Western blot for detecting overexpressed and recombinant human Pax3 protein, but may not be suitable for detecting endogenous Pax3.

By immunofluorescence, PA1-107 has been used to stain frozen embryonic mouse brain tissue.

PA1-107 has been validated for IP using overexpressed Pax3 protein.

Target Information

Pax 3 (Paired Box 3) is a transcription factor that is involved in development of the peripheral nervous system, melanocytes, some vascular smooth muscle, and a number of other derivatives. It regulates neurogenesis in pre-migratory neural crest cells from the dorsal neural tube, and in myogenic progenitors in the presomitic mesoderm and the hypaxial somites. Alternative splicing results in isoforms having different C-termini.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: HuP2; MGC120381; MGC120382; MGC120383; MGC120384; MGC134778; paired box gene 3; paired box homeotic gene 3; Paired box protein Pax-3; paired domain gene 3; paired domain gene HuP2; PAX3/FKHR fusion

Gene Aliases: CDHS; HUP2; Pax-3; PAX3; Sp; splotch; WS1; WS3

UniProt ID: (Human) P23760, (Mouse) P24610

Entrez Gene ID: (Human) 5077, (Mouse) 18505

Function(s)