- PA1-100X - Invitrogen Antibodies GATA2 antibody

Submitted references Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear.

Burns JC, Kelly MC, Hoa M, Morell RJ, Kelley MW
Nature communications 2015 Oct 15;6:8557

Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition.

Lizama CO, Hawkins JS, Schmitt CE, Bos FL, Zape JP, Cautivo KM, Borges Pinto H, Rhyner AM, Yu H, Donohoe ME, Wythe JD, Zovein AC
Nature communications 2015 Jul 23;6:7739

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of GATA2 was performed by loading 100 ng of GST-tagged human GATA2 and GATA1 recombinant proteins and 10 µL PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288) and blocked with SuperBlock T20 blocking buffer (Product # 37536) in TBST for 1 hour at room temperature. GATA2-GST fusion ptrotein was detected at ~80 kD using an GATA2 rabbit polyclonal antibody (Product # PA1-100) (left panel), stripped, and reprobed with GATA1 and GATA2 rabbit polyclonal antibodies (middle panel). Blots were stripped again and reprobed with a GST-tag (right panel) polyclonal antibody. All antibodies were used at a dilution of 1:1000 in blocking buffer overnight at 4°C or for 1 hour at room temperature on a rocking platform, followed by a Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # 31460) at a dilution of 1:20,000 for 1 hour at room temperature. Recombinant GST-fusion protein breakdown bands are indicated in the right panel. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). Images were acquired on a Thermo Scientific myECL Imager (Product # 62236).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of GATA2 was performed by loading 25 µg of either MEF or NTERRA whole cell lysate 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. Membranes were then probed with a rabbit polyclonal antibody recognizing GATA2 (Product # PA1-100) at a dilution of 1:500 overnight at 4°C on a rocking platform. Membranes were then washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of GATA2 (green) in Hela cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a GATA2 polyclonal antibody (Product # PA1-100) at a dilution of 1:200 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of GATA2 (green) in U251 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a GATA2 polyclonal antibody (Product # PA1-100) at a dilution of 1:20 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of GATA2 (green) in human cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a GATA2 polyclonal antibody (Product # PA1-100) at a dilution of 1:100 and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Images were taken at a 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of GATA2 (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were then blocked with 5% normal goat serum (Product # 31873) for 15 minutes at room temperature. Cells were then probed with a rabbit polyclonal antibody recognizing GATA2 (Product # PA1-100) at a dilution of 1:200 for at least 1 hour at 37°C. Cells were then washed with PBS and incubated with DyLight 488 goat-anti-rabbit secondary antibody at a dilution of 1:200 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 10X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on human prostate tissue. To expose target protein, antigen was retreived using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a GATA2 Rabbit polyclonal antibody (Product # PA1-100) at a dilution of 1:100 overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 SOX17 directly binds Runx1 and Gata2 for repression of haematopoietic fate. ( a ) SOX17 chromatin immunoprecipitation (ChIP) qRT-PCR of E11.0 sorted endothelial cells. Letters denote regions with SOX17-binding site consensus sequences upstream of Runx1 and Gata2 promoters, and Lef1 as a positive control. Error bars indicate s.e.m. IgG control set to one for comparisons of fold change, n =3 litters, embryos pooled, P values as indicated. ( b ) Electrophoretic mobility shift assay (EMSA) of putative SOX17-binding sites within ChIP sequences designated by letters in a . Each lane represents biotin-labelled duplexed oligonucleotides containing the Lef1 promoter SOX17-binding site (Lef1_Biot). Addition of rSOX17-Flag produces a specific shift, indicating protein-DNA complex (lane 2), which is competed away by unlabelled Lef1 (Lef1_s1), while mutant probe does not compete (Lef1_Delta_s1). Similar designations are used for putative binding sites (and mutants) in Runx1 and Gata2 sequences. Asterisks denote competitive binding. ( c ) Bar graph depicts luciferase activity of Gata2 and Runx1 promoters after Sox17 siRNA versus control (scramble). P values as indicated. Error bars represent s.e.m. ( d ) Immunofluorescence of haematopoietic cell clusters (arrowhead) in E10.5 dorsal aorta (DA) of Sox17 f/f and Sox17 f/+ mutants (after tamoxifen mediated Cre induction at E9.5). Traced cells labelled in red (Td + ), SOX17 in grey and RUNX1 in green. DAPI in blue. Scale bar, 10 mum. S

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Single-cell RNA-Seq identifies unique cell types and novel markers in the newborn mouse utricle. ( a ) K -means clustering of 158 P1 utricle cells ( x axis) using the top 195 genes ( y axis) identified with PCA. The gap statistic, an unbiased estimate of the total number of clusters, identified seven distinct clusters. On the basis of expression of known marker genes and fluorescence state (bottom x -axis bars and right legends, displayed on log 2 scale), each cluster was assigned to one of the three known cell types (top x -axis bars): TECs (grey), SCs (light green) and HCs (light red). TECs comprise one cluster, whereas SCs and HCs comprise two (SC.i-ii) and four (HC.i-iv) clusters, respectively. Genes were also divided by k -means (black bars indicate 15 clusters identified by gap statistic) and pooled based on cell type, HC maturity or variation across multiple cell types (Mixed). The Mixed group consists of two gene clusters expressed in non-HCs and one small cluster restricted to SCs and HCs. ( b ) Violin plots for representative genes identified as differentially expressed (FDR

PA1-100X

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