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<strong>Figure 1 Western Blot Validation of PUMA in K562 Cells</strong><br>Loading: 15 μg of lysates per lane.Antibodies: 3043 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Human Cells</strong><br>Loading: 20 μg of lysates per lane.Antibodies: 3041 (3 μg/mL), 3043 (2 μg/mL), beta-actin (1 μg/mL) and GAPDH (0.02 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Immunofluorescence Validation of PUMA in K562 Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3043 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red) and DAPI staining (blue).
<strong> Figure 4 Immunohistochemistry Validation of PUMA in Human Breast Carcinoma </strong><br> Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-PUMA antibody (3043) at 10 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong> Figure 5 Immunohistochemistry Validation of PUMA in Human Breast Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded human breast tissue using anti-PUMA antibody (3043) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 6 Immunofluorescence Validation of PUMA in K562</strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3043 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing cytosol staining on K562 cells
<strong>Figure 7 KO Validation of PUMA in Mouse Thymocytes (Michalak et al., 2008) </strong><br>Western blot analysis ofthymocytes from wt, noxa knockout, puma knockout and noxa/puma double knockout mice cultured for 7 h in the presence or absence of 2.5 Gy g-irradiation.PUMA expression was not detected in puma KO and double KO mice with anti-PUMA antibodies (3043).
<strong>Figure 8 KO Validation of PUMA in Mouse Cerebellar Neurons (Ambacher et al., 2012) </strong><br>Puma expression is induced by potassium withdrawal in cerebellar granule neurons. After 7 days in culture CGNs were either maintained in media containing 25 mM potassium (K25) or switched to low potassium medium containing 5 mM potassium (K5). PUMA protein levels were analyzed by western blot with anti-PUMA antibodies (3043). PUMA expression was not detected in PUMA KO mice and was increased after treatment in WT.
<strong>Figure 9 Induced Expression of PUMA in MCF7 cells (Wade et al., 2008) </strong><br>Western analysis of MCF7 treated with the indicated dose of Nutlin-3a orABT-737 for 24h. Note that Puma is induced following Nutlin-3a treatment in these cells and PUMA expression was detected by anti-PUMA antibodies (3043)
<strong>Figure 10 Immunofluorescence Validation of PUMA in Rat Retina (Wakabayashi et al., 2012) </strong><br>PUMA expression in the rat retina detected by anti-PUMA antibodies (3043). The specimens were counterstained with Hoechst 33258 to visualize nuclei (+DNA). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; P, postnatal day.
Figure 1 Western Blot Validation of PUMA in K562 Cells
Loading: 15 μg of lysates per lane.Antibodies: 3043 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution
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IAV
KO
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PUMA Antibody

CATALOG NUMBER: 3043

Clonality:
Polyclonal
Tested Applications:
ELISA, IF, IHC-P, WB
Host Species:
Rabbit
Species Reactivity:
Human, Rat
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human, Rat
Homology:
Predicted species reactivity based on immunogen sequence: Mouse: (78.6%)
Immunogen:
Anti-PUMA antibody (3043) was raised against a peptide corresponding to 14 amino acids near the amino terminus of human PUMA isoform 1.
The immunogen is located within the first 50 amino acids of PUMA.
Conjugate:
Unconjugated
Tested Applications:
ELISA, IF, IHC-P, WB
Application Note:
WB: 2-3 μg/mL; IF: 10-20 μg/mL; IHC: 2.5-10 μg/mL.
Antibody validated: Western Blot in human samples; Immunohistochemistry in human samples; Immunofluorescence in human samples. All other applications and species not yet tested.
Positive Control 1:
Cat. No. 1210 - 293 Cell Lysate
Positive Control 2:
Cat. No. 1211 - HepG2 Cell Lysate
Positive Control 3:
Cat. No. 10-001 - Human Breast Tissue Slide
Positive Control 4:
Cat. No. 1204 – K562 Cell Lysate
Predicted Molecular Weight:
Predicted: 21kD
Observed: 23kD
Validation:

Independent Antibody Validation (Figure 2) shows similar PUMA expression profile in both human and mouse cell lines detected by two independent anti-PUMA antibodies that recognize different epitopes, 3041 against C-terminus and 3043 against the N-terminus.  PUMA proteins are detected in most of the cell lines with different expression levels by the two independent antibodies.  

KO Validation (Figure 7, 8) shows lack of PUMA expression in the thymocytes or neurons of PUMA knockout mice.

Animal Species Reactivity  (Figure 10) shows PUMA expression in the rat retina detected with anti-PUMA antibodies (3043).

Isoforms:
Human PUMA has 4 isoforms, including isoform 1 (193aa, 21kD), isoform 2 (131aa, 14kD), isoform 3 (101aa, 10kD) and isofrom 4 (261aa, 27kD). This antibody detects human isoform 1, and is predicted to detect mouse and rat PUMA (193aa, 21kD for both of them).
Purification:
PUMA Antibody is affinity chromatography purified via peptide column.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
PUMA Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
PUMA antibody can be stored at 4°C for three months and -20°C, stable for up to one year. As with all antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures.
Ncbi Official Symbol:
BBC3
Additional Names:
PUMA Antibody: JFY1, PUMA, JFY-1, Bcl-2-binding component 3
Protein Accession Number:
NP_055232
Protein Gi Number:
15193488
Ncbi Gene Id Number:
27113
User Note:
Optimal dilutions for each application to be determined by the researcher.
Background:
PUMA Antibody: Apoptosis is related to many diseases and development. The p53 tumor-suppressor protein induces apoptosis through transcriptional activation of several genes. A novel p53 inducible pro-apoptotic gene was identified recently and designated PUMA (for p53 upregulated modulator of apoptosis) and bbc3 (for Bcl-2 binding component 3) in human and mouse. PUMA/bbc3 is one of the pro-apoptotic Bcl-2 family members including Bax and Noxa, which are also transcriptional targets of p53 (1). The PUMA gene encodes two BH3 domain-containing proteins termed PUMA-alpha and PUMA-beta (2). PUMA proteins bind Bcl-2, localize to the mitochondria, and induce cytochrome c release and apoptosis in response to p53. PUMA may be a direct mediator of p53-induced apoptosis.
Background Reference 1:
Nakano and Vousden. Mol Cell. 2001; 7(3)683-94
Background Reference 2:
Han et al. Proc Natl Acad Sci U S A. 2001;98(20):11318-23.
Citation 1:
Michalak et al. In several cell types tumour suppressor p53 induces apoptosis largely via Puma but Noxa can contribute. Cell Death Differ. 2008;15(6):1019-29. PMID: 18259198
Citation 2:
Ambacher et al. The JNK- and AKT/GSK3β- signaling pathways converge to regulate Puma induction and neuronal apoptosis induced by trophic factor deprivation. PLoS One. 2012;7(10): e46885. PMID: 23056511
Citation 3:
Wade et al. BH3 activation blocks Hdmx suppression of apoptosis and cooperates with Nutlin to induce cell death. Cell Cycle. 2008;7(13):1973-82. PMID: 18604177
Citation 4:
Wakabayashi et al. Prolonged expression of Puma in cholinergic amacrine cells during the development of rat retina. J Histochem Cytochem. 2012;60(10):777-88. PMID: 22736709
Citation 5:
Charvet et al. Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53. Mol Cell. 2011;42(5):584-96. PMID: 21658600
Citation 6:
Schubert et al. Requirement of GSK-3 for PUMA induction upon loss of pro-survival PI3K signaling. Cell Death Dis. 2018;9(5):470. PMID: 29686375
Citation 7:
Jain et al. A phase I-II trial of fludarabine, bendamustine and rituximab (FBR) in previously treated patients with CLL. Oncotarget. 2017;8(13):22104-12. PMID: 27655665
Citation 8:
Ikonomopoulou et al. Gomesin inhibits melanoma growth by manipulating key signaling cascades that control cell death and proliferation. Sci Rep. 2018 ;8(1):11519. PMID: 30068931

FOR RESEARCH USE ONLY

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Disclaimer:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.

CATALOG NUMBER:

3043

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

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