SOX2 Monoclonal Antibody (20G5) Catalog Number MA1-014 Product data sheet

Transcription

1 Website: thermofisher.com Customer Service (US): ext. 1 Technical Support (US): ext. 441 SOX2 Monoclonal Antibody (20G5) Catalog Number MA1-014 Product data sheet Details Size 100 µg Host/Isotope Class Type Clone Immunogen Conjugate Form Concentration Purification Storage buffer Contains Storage Conditions Mouse / IgG1, kappa Monoclonal Antibody 20G5 Full-length human recombinant protein expressed in bacteria Unconjugated Liquid 1 mg/ml Protein A PBS with 30% glycerol, 1mg/mL BSA 0.05% sodium azide -20 C Species Reactivity Species reactivity Published species Tested Applications Dilution * ChIP assay (ChIP) 1-3 µl Flow Cytometry (Flow) 1:100 Immunocytochemistry (ICC) 1:200 Immunofluorescence (IF) 1:200 Immunohistochemistry (Paraffin) (IHC (P)) Immunoprecipitation (IP) 5 µg Dog, Fish, Human, Mouse Human, Not Applicable 1:20-1:200 Western Blot (WB) 1:1000-1:2000 Published Applications Miscellaneous PubMed (MISC) Immunohistochemistry (IHC) See 1 publications below See 1 publications below * Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls. Product specific information Western blot analysis of MA1-014 specifically detects SOX2 protein at ~36 kda in the nucleus of human and mouse embryonal carcinoma and ips cells. Background/Target Information SOX2 is an intronless gene encoding a member of the SRY-related HMG-box (SOX) family of transcription factors involved in the regulation of embryonic development and in the determination of cell fate. The product of the SOX2 gene is required for stem-cell maintenance in the central nervous system, and also regulates gene expression in the stomach. The SOX2 gene lies within an intron of another gene called SOX2 overlapping transcript (SOX2OT). Further, SOX2 protein may act as a transcriptional activator after forming a protein complex with other proteins. Mutations in the SOX2 gene have been associated with bilateral anophthalmia, a severe form of structural eye malformation, optic nerve hypoplasia and syndromic microphthalmia.

2 Advanced Verification Data SOX2 Antibody (MA1-014) The specificity of anti-sox2 monoclonal antibody (Product # MA1-014) was demonstrated using chromatin immunoprecipitation (ChIP). q-pcr was used to demonstrate levels of enrichment of SOX2 at the expected loci in exon 1 or exon-2-3 of EGR1 gene but not negative site in -15kb region of the same gene in HTC-IR rat hepatoma cells treated with insulin for 10 minutes compared to those in untreated cells. Cell Treatment validation info. Product Images For SOX2 Monoclonal Antibody (20G5) Immunofluorescent analysis of Sox2 (green) in H9 embryonic stem cells grown for a few days on Matrigel-coated chamber slides. Cells fixed in 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Cells were probed with a Sox2 monoclonal antibody (Product # MA1-014) at a dilution of 1:200 overnight at 4 C, washed with PBST, and incubated with a fluorescein-conjugated secondary antibody at a dilution of 1:100 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI and cells were analyzed by fluorescence microscopy at 20X magnification. SOX2 Antibody (MA1-014) in IHC (P) Immunohistochemistry analysis of SOX2 showing staining in the nucleus of paraffin-treated human lung squamous carcinoma (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (ph 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddh2o and PBS, and then probed with a SOX2 monoclonal antibody (Product # MA1-014) diluted by 3% BSA-PBS at a dilution of 1:200 overnight at 4 C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

3 SOX2 Antibody (MA1-014) in IP Immunoprecipitation of Sox2 was performed on NCCIT cells. Antigen-antibody complexes were formed by incubating 500 µg of NCCIT whole cell lysate with 5 µg of a Sox2 monoclonal antibody (Product # MA1-014) overnight on a rocking platform at 4 C. The immune complexes were captured on 50 µl Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Eluted sample and 25 µg of NCCIT whole cell lysate (loading control) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween-20 for at least 1 hour. The membrane was probed with a Sox2 monoclonal antibody (Product # MA1-014) at a dilution of 1:1000 overnight rotating at 4 C, washed in TBST, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 32430) at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). SOX2 Antibody (MA1-014) in WB Western blot analysis of Sox2 was performed by loading 25 µg of various whole cell lysates per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Sox2 monoclonal antibody (Product # MA1-014) at a dilution of 1:1500 overnight at 4 C on a rocking platform, washed in TBS-0.1% Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 32430) at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). SOX2 Antibody (MA1-014) in Flow Flow cytometric analysis of Sox2 (blue histogram) on HEL 11.4 induced IPS cells. To generate single cells suspensions, colonies were treated with TrypLE cell dissociation enzyme for 5 minutes at 37 C. Cells were incubated with a Sox2 monoclonal antibody (Product # MA1-014) or mouse IgG (green histogram) at a dilution of 1:100 for 1 hour on ice, washed with PBS + 5% fetal calf serum (FACS buffer), and incubated with a fluorescein-conjugated secondary antibody at a dilution of 1:200 for 30 minutes on ice. Cells were washed with cold FACS buffer, resuspended in 500 µl of FACS buffer containing 10 µl of 4% paraformaldehyde, and analyzed on a flow cytometer. SOX2 Antibody (MA1-014) in ChIP Chromatin immunoprecipitation analysis of SOX2 was performed using cross-linked chromatin from 1x10^6 HTC-IR rat hepatoma cells treated with insulin for 0, 10, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: with 1.0 µl/100 µl well volume of a SOX2 monoclonal antibody (Product # MA1-014). Chromatin aliquots from ~1x10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µl of eluted DNA in 2 µl SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 or exon-2-3 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the rat Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.

4 Immunofluorescent analysis of Sox2 (green) in human neural stem cells derived from PD-3 ipscs using Gibco PSC Neural Induction Medium (Product # A ). 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 Sox2 monoclonal antibody (Product # MA1-014) at a dilution of 1:200 in blocking buffer for 3 hours at room temperature, and then incubated with an DyLight 488- conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:250 for 1 hour at room temperature. Image was taken on an EVOS FLoid Cell Imaging Station at 10X magnification. Immunofluorescent analysis of SOX2 (red) in human ipsc-derived forebrain organoids derived at Day 40. The organoids were fixed with 4% PFA for 1 hour at room temperature, followed by incubation with 30% sucrose solution overnight at 4 C. The organoids were then embedded in OCT and cryosectioned at 5 µm, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were stained with a Mouse SOX2 monoclonal antibody (red; Product # MA1-014) at a dilution of 1:500 in blocking buffer overnight at 4 C, and then incubated with Donkey anti-mouse Alexa Fluor 568 (Product # A10037) at a dilution of 1:1000 in blocking solution at room temperature for 1 hour. Images were taken at 20X magnification. Scale bar: 50 µm. Data courtesy of Dr. Zhexing Wen at Emory University. Immunofluorescent analysis of Sox2 (green) in HEL 11.4 induced IPS cells grown for a few days on Matrigel-coated chamber slides. Cells fixed in 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Cells were probed with a Sox2 monoclonal antibody (Product # MA1-014) at a dilution of 1:200 overnight at 4 C, washed with PBST, and incubated with a fluorescein-conjugated secondary antibody at a dilution of 1:100 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI and cells were analyzed by fluorescence microscopy at 20X magnification. SOX2 Antibody (MA1-014) in IHC (P) Immunohistochemistry was performed on deparaffinized canine fetal brain tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (ph 6.0) buffer for 20 minutes at 95 C. Following antigen retrieval tissues were blocked with Avidin/Biotin Blocking kit (Product # ) at room temperature and then probed with a Sox2 monoclonal antibody (Product # MA1-014) at a dilution of 1:200 for one hour at room temperature. Tissues were washed extensively with TBS % Triton X-100 (Product # 28314). Detection was performed using a goat antimouse HRP secondary antibody (Product # 31430) at a dilution of 1:500 followed by colorimetric detection using metal enhanced DAB (Product # 34065). Tissues were counterstained with hematoxylin and prepped for mounting. Images were taken on a Zeiss Axiovision microscope at 20X magnification. The bottom layer with a dense staining of Sox2 is in the subependymal plate, where there are large numbers of progenitor cells. The upper layer is neuroparenchyma where cells are differentiated, therefore less staining of Sox2. Note: Data courtesy of Innovators Program.

5 SOX2 Antibody (MA1-014) in IHC (P) Immunohistochemistry analysis of SOX2 showing staining in the nucleus of paraffin-treated mouse esophagus tissue (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (ph 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddh2o and PBS, and then probed with a SOX2 monoclonal antibody (Product # MA1-014) diluted by 3% BSA-PBS at a dilution of 1:20 overnight at 4 C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting. SOX2 Antibody (MA1-014) in IHC (P) Immunohistochemistry analysis of SOX2 showing staining in the nucleus of paraffin-treated human glioma tissue (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (ph 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddh2o and PBS, and then probed with a SOX2 monoclonal antibody (Product # MA1-014) diluted by 3% BSA-PBS at a dilution of 1:200 overnight at 4 C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting. SOX2 Antibody (MA1-014) in WB Western blot analysis of SOX2 was performed by loading 50 µg of F9 and negative control NIH3T3 whole cell lysates, and 10 µl of PageRuler Prestained Protein Ladder (Product # 26616) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a SOX2 monoclonal antibody (Product # MA1-014) at a dilution of 1:1000 overnight at 4 C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-mouse HRP secondary antibody (Product # 32430) at a dilution of 1: 20,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). SOX2 Antibody (MA1-014) in Flow Flow cytometric analysis of Sox2 (blue histogram) on H9 embryonic stem cells. To generate single cells suspensions, colonies were treated with TrypLE cell dissociation enzyme for 5 minutes at 37 C. Cells were incubated with a Sox2 monoclonal antibody (Product # MA1-014) or mouse IgG (green histogram) at a dilution of 1:100 for 1 hour on ice, washed with PBS + 5% fetal calf serum (FACS buffer), and incubated with a fluorescein-conjugated secondary antibody at a dilution of 1:200 for 30 minutes on ice. Cells were washed with cold FACS buffer, resuspended in 500 µl of FACS buffer containing 10 µl of 4% paraformaldehyde, and analyzed on a flow cytometer.

6 SOX2 Antibody (MA1-014) in Flow Flow cytometry analysis of Sox2 on human neural stem cells derived from PD-3 ipscs using Gibco PSC Neural Induction Medium (Product # A ). Cells were fixed, permeabilized and stained with a Sox2 monoclonal antibody (Product # MA1-014, red histogram) or mouse IgG1 isotype control (black histogram) at a 1:100 dilution in 5% BSA. After incubation of the primary antibody for 1 hour on ice, the cells were stained with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:500 for 1 hour on ice. A representative 10,000 cells were acquired for each sample.

7 PubMed References For SOX2 Monoclonal Antibody (20G5) 1 Miscellaneous PubMed References Species / Dilution Human / 1:1000 Summary 1 Immunohistochemistry References Species / Dilution MA1-014 was used in western blot to discuss Phage lambda integrase as a transgenesis tool Nucleic acids research (Apr 2016; 44: null) "Conservative site-specific and single-copy transgenesis in human LINE-1 elements." Author(s):Vijaya Chandra SH,Makhija H,Peter S,Myint Wai CM,Li J,Zhu J,Ren Z,D'Alcontres MS,Siau JW,Chee S,Ghadessy FJ,Dröge P PubMed Article URL: Summary MA1-014 was used in immunohistochemistry to analyze triploid breast cancers non-responsive to neoadjuvant therapy by study of aneuploidy and senescence paradoxes Not Applicable / 1:50 Histochemistry and cell biology (Apr 2016; 145: 497) "Disentangling the aneuploidy and senescence paradoxes: a study of triploid breast cancers non-responsive to neoadjuvant therapy." Author(s):Gerashchenko BI,Salmina K,Eglitis J,Huna A,Grjunberga V,Erenpreisa J PubMed Article URL: