PA1-806

antibody from Invitrogen Antibodies

Targeting: FOXP3 AIID, DIETER, IPEX, JM2, PIDX, SCURFIN, XPID

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

PA1-806

Provider

Invitrogen Antibodies

Product name

FOXP3 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

PA1-806 detects FOXP3 from human samples. PA1-806 has been successfully used in Western blot and ChIP procedures. By Western blot, PA1-806 detects a ~50 kDa band representing FOXP3 from Hela cells. The PA1-806 immunogen is a synthetic peptide corresponding to residues W(406) T V D E L E F R K K R S Q R(420) of human FOXP3. PA1-806 can be used with blocking peptide PEP-297.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

1 mg/mL

Storage

-20° C, Avoid Freeze/Thaw Cycles

References

Submitted references

MEF2D sustains activation of effector Foxp3+ Tregs during transplant survival and anticancer immunity.

Di Giorgio E, Wang L, Xiong Y, Akimova T, Christensen LM, Han R, Samanta A, Trevisanut M, Bhatti TR, Beier UH, Hancock WW
The Journal of clinical investigation 2020 Dec 1;130(12):6242-6260

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (20 µg lysate) of HeLa (Lane 1), K562 (Lane 2), Jurkat (Lane 3), MCF7 (lane 4), Hep G2 (lane 5), PC-3 (lane 6), COLO 205 (lane 7), HT-29 (lane 8) and HCT 116 (lane 9). The blots were probed with Anti-FOXP3 Rabbit Polyclonal Antibody (Product # PA1-806, 1:250-1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 50 kDa band corresponding to FOXP3 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of FOXP3 was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with FOXP3 Rabbit Polyclonal Antibody (Product # PA1-806) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of FOXP3 was done on Jurkat cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with FOXP3 Rabbit Polyclonal Antibody (PA1806, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Chromatin immunoprecipitation analysis of FOXP3 was performed using cross-linked chromatin from 1x10^6 HCT116 colon carcinoma cells treated with serum for 0, 15, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a FOXP3 polyclonal antibody (Product # PA1-806). 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 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 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.