PUMA Antibody

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ProSci
Product Code: PSI-3041
Product Group: Primary Antibodies
Supplier: ProSci
CodeSizePrice
PSI-3041-0.02mg0.02mg£150.00
Quantity:
PSI-3041-0.1mg0.1mg£449.00
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Overview

Host Type: Rabbit
Antibody Isotype: IgG
Antibody Clonality: Polyclonal
Regulatory Status: RUO
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunofluorescence (IF)
  • Immunohistochemistry (IHC)
  • Western Blot (WB)

Images

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<strong>Figure 1 Western Blot Validation of PUMA in K562 and 3T3/NIH Cells</strong><br> Loading: 15 μg of lysates per lane. Antibodies: 3041 (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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<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</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.
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<strong>Figure 3 Validation with PUMA siRNA Knockdown in 293 Cells</strong><br> 293 cells were transfected with control siRNAs (lane 1) or PUMA siRNAs (lane 2) Loading: 15 μg of 293 whole cell lysates per lane. Antibodies: 3041 (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.
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<strong>Figure 4 Sensitivity Test for PUMA in 2983 Cells</strong><br> Loading: Lysates/proteins at 15 μg per lane. Antibodies: 3041 (lane 1-3: 1, 2 and 4 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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<strong>Figure 5 Immunofluorescence Validation of PUMA in K562 Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3041 at 2 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing cytosol staining on K562 cells.
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<strong> Figure 6 Immunocytochemistry Validation of PUMA in K562 Cells </strong><br> Immunocytochemical analysis of K562 cells using anti-PUMA antibody (3041) at 1 μg/ml. Cells 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.
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<strong>Figure 7 KD Validation of PUMA in HOSE-RasV12 Cells (Elgendy et al., 2011) </strong><br> HOSE-RasV12 cells were transfected with control shRNA plasmid or shRNA plasmids (KD) targeted against Noxa or Puma, as indicated. PUMA expression was not observed in PUMA KD cells detected by anti-PUMA antibodies (3041).
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<strong>Figure 8 Induction Validation of PUMA in Primary Cortical Neurons (Sabirzhanov et al., 2014) </strong><br> PUMA protein levels were increased in etoposide-treated primary cortical neurons detected by anti-PUMA antibodies (3041).
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<strong>Figure 9 KD Validation of PUMA PUMA in Tet Cells (Han et al., 2010) </strong><br> Immunoblot analyses of Tet-induced p53 cells treated with NOXA, Puma, Bim or non-targeting siRNAs that were utilized in this experiment. PUMA protein levels were markedly reduced in PUMA KD cells detected by anti-PUMA antibodies (3041).
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<strong>Figure 1 Western Blot Validation of PUMA in K562 and 3T3/NIH Cells</strong><br> Loading: 15 μg of lysates per lane. Antibodies: 3041 (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 Cell Lines</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 Validation with PUMA siRNA Knockdown in 293 Cells</strong><br> 293 cells were transfected with control siRNAs (lane 1) or PUMA siRNAs (lane 2) Loading: 15 μg of 293 whole cell lysates per lane. Antibodies: 3041 (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 4 Sensitivity Test for PUMA in 2983 Cells</strong><br> Loading: Lysates/proteins at 15 μg per lane. Antibodies: 3041 (lane 1-3: 1, 2 and 4 μg/mL). 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunofluorescence Validation of PUMA in K562 Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 cells labeling PUMA with 3041 at 2 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing cytosol staining on K562 cells.
<strong> Figure 6 Immunocytochemistry Validation of PUMA in K562 Cells </strong><br> Immunocytochemical analysis of K562 cells using anti-PUMA antibody (3041) at 1 μg/ml. Cells 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 7 KD Validation of PUMA in HOSE-RasV12 Cells (Elgendy et al., 2011) </strong><br> HOSE-RasV12 cells were transfected with control shRNA plasmid or shRNA plasmids (KD) targeted against Noxa or Puma, as indicated. PUMA expression was not observed in PUMA KD cells detected by anti-PUMA antibodies (3041).
<strong>Figure 8 Induction Validation of PUMA in Primary Cortical Neurons (Sabirzhanov et al., 2014) </strong><br> PUMA protein levels were increased in etoposide-treated primary cortical neurons detected by anti-PUMA antibodies (3041).
<strong>Figure 9 KD Validation of PUMA PUMA in Tet Cells (Han et al., 2010) </strong><br> Immunoblot analyses of Tet-induced p53 cells treated with NOXA, Puma, Bim or non-targeting siRNAs that were utilized in this experiment. PUMA protein levels were markedly reduced in PUMA KD cells detected by anti-PUMA antibodies (3041).

Documents

Further Information

Additional Names:
PUMA Antibody: JFY1, PUMA, JFY-1, Bcl-2-binding component 3
Application Note:
WB: 1-4 μg/mL; IF: 2 μg/mL; ICC: 1 μg/mL.

Antibody validated: Western Blot in human and mouse samples; Immunocytochemistry and Immunofluorescence in human samples. All other applications and species not yet tested.
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 References:
  • Nakano and Vousden. Mol Cell. 2001; 7(3)683-94
  • Han et al. Proc Natl Acad Sci U S A. 2001;98(20):11318-23.
Buffer:
PUMA Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Conjugate:
Unconjugated
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.
Homology:
Predicted species reactivity based on immunogen sequence: Rat: (100%)
Immunogen:
Anti-PUMA antibody (3041) was raised against a peptide corresponding to 14 amino acids near the carboxyl terminus human PUMA isoform 1.

The immunogen is located within the last 50 amino acids of PUMA.
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&2, mouse and rat PUMA (193aa, 21kD for both of them).
NCBI Gene ID #:
27113
NCBI Official Name:
BCL2 binding component 3
NCBI Official Symbol:
BBC3
NCBI Organism:
Homo sapiens
Physical State:
Liquid
PREDICTED MOLECULAR WEIGHT:
23 kDa
Protein Accession #:
NP_055232
Protein GI Number:
15193488
Purification:
PUMA Antibody is affinity chromatography purified via peptide column.
Research Area:
Apoptosis,Neuroscience,Autophagy,Cancer
SPECIFICITY:
A lower band at approximately 16 kDa was detected in Daudi and K562 cells, which may represent the PUMA isoform 2.
Swissprot #:
Q96PG8
User NOte:
Optimal dilutions for each application to be determined by the researcher.
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.  

siRNA Knockdown Validation (Figure 3): Anti-PUMA antibody (3041) specificity was further verified by PUMA specific siRNA knockdown. PUMA signal in HeLa cells transfected with PUMA siRNAs was much weaker in comparison with that in HeLa cells transfected with control siRNAs.

References

  1. Elgendy et al. Oncogenic Ras-induced expression of Noxa and beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol. Cell. 2011;42(1):23-35. PMID: 21353614
  2. Han et al. Regulation of mitochondrial apoptotic events by p53-mediated disruption of complexes between anti-apoptotic Bcl-2 members and Bim. J.B.C. 2010; 285(29):22473-83. PMID: 20404322
  3. Sabirzhanov et al. Downregulation of miR-23a and miR-27a following experimental traumatic brain injury induces neuronal cell death through activation of proapoptotic Bcl-2 proteins. J Neurosci. 2014;34(30):10055-71. PMID: 25057207
  4. O'Neill et al. Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane. Genes Dev. 2016;30(8):973-88. PMID: 27056669
  5. Sabirzhanov et al. miR-711 upregulation induces neuronal cell death after traumatic brain injury. Cell Death Differ. 2016;23(4):654-68. PMID: 26470728
  6. Shi et al. NSC-87877 inhibits DUSP26 function in neuroblastoma resulting in p53-mediated apoptosis. Cell Death Dis. 2015;6:e1841. PMID: 26247726
  7. Meng et al. Dose-response transition from cell cycle arrest to apoptosis with selective degradation of Mdm2 and p21WAF1/CIP1 in response to the novel anticancer agent, aminoflavone (NSC 686,288). Oncogene. 2007;26(33):4806-16. PMID: 17297446
  8. Lee and Pelletier. Dependence of p53-deficient cells on the DHX9 DExH-box helicase. Oncotarget. 2017;8(19):30908-30921. PMID: 28427210

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