MA3-414

antibody from Invitrogen Antibodies

Targeting: PTGES3 cPGES, p23, TEBP

Provider product page for MA3-414

Western blot

Antibody data

Antibody Data
Antigen structure
References [42]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [4]
Immunohistochemistry [3]
Flow cytometry [3]
Other assay [15]

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Product number: MA3-414 - Provider product page
Provider: Invitrogen Antibodies
Product name: p23 Monoclonal Antibody (JJ3)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA3-414 detects p23 from chicken, guinea pig, human, rat, mouse and rabbit samples.
Antibody clone number: JJ3
Concentration: Conc. Not Determined

PTGES3 protein structure - MA3-414 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of p23 was performed by loading 25 µg of Hela (Lane 1), HepG2 (Lane 2), and mouse spleen cell lysates (Lane 3) and a molecular weight protein ladder onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with a blocking buffer at 4ºC overnight. The membrane was probed with a p23 monoclonal antibody (Product # MA3-414) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at 23 kDa in all three cell lines.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of p23 was achieved by transfecting T-47D with p23 specific siRNAs (Silencer® select Product # S21075 and S21074). Western blot analysis (Fig. a) was performed using Whole cell extracts from the p23 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with p23 Monoclonal Antibody (JJ3) (Product # MA3-414, 1:2000 dilution ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to p23.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-p23 Monoclonal Antibody (JJ3) (Product # MA3-414) and a 18 kDa band corresponding to p23 was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of T-47D (Lane 1), HeLa (Lane 2), NIH/3T3 (Lane 3), L6 (Lane 4); and tissue extracts Mouse Spleen (Lane 5) and Rat Spleen (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of p23 using Anti-p23 Monoclonal Antibody (JJ5) (Product # MA3-414) shows staining in C6 Cells. p23 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing p23 (Product # MA3-414) at a dilution of 1:500 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of p23 using Anti-p23 Monoclonal Antibody (JJ5) (Product # MA3-414) shows staining in Hela Cells. p23 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing p23 (Product # MA3-414) at a dilution of 1:500 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of p23 was performed using 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 2% BSA for 45 minutes at room temperature. The cells were labeled with p23 Monoclonal Antibody (JJ3) (Product # MA3-414) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Product # A32787), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Blue) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nucleus and cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60x magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of p23 using Anti-p23 Monoclonal Antibody (JJ5) (Product # MA3-414) shows staining in MCF-7 Cells. p23 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing p23 (Product # MA3-414) at a dilution of 1:500 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human breast carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:1000 with a mouse monoclonal antibody recognizing p23 (Product # MA3-414) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human testis tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:1000 with a mouse monoclonal antibody recognizing p23 (Product # MA3-414) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:1000 with a mouse monoclonal antibody recognizing p23 (Product # MA3-414) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of p23 in 3T3 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with p23 monoclonal antibody (Product # MA3-414) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of p23 in Hela cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with p23 monoclonal antibody (Product # MA3-414) at a dilution of 0.25 µg/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of p23 in Jurkat cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with p23 monoclonal antibody (Product # MA3-414) at a dilution of 0.25 µg/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: RNA immunoprecipitation (RIP) western of p23 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of p23 monoclonal antibody (Product # MA3-414) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a p23 monoclonal antibody (Product # MA3-414) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4. Caspase-1 exhibits greater promiscuity toward natural substrates than caspases-3 or -7. A , processing of substrate proteins in Jurkat cell-free extracts after addition of the indicated amounts of recombinant caspase ( Casp )-1, -3, and -7 followed by incubation for 2 h at 37 degC. Recombinant caspases were active site titrated (see supplemental Fig. S1 ) as described under ""Experimental Procedures."" Recombinant IL-1beta (10 ng per reaction) was added to cell-free extracts as a positive control for caspase-1 activity. Substrate proteolysis was detected by SDS-PAGE followed by immunoblotting. B , caspase-1-mediated processing of the purified recombinant proteins over the indicated concentration ( Conc. ) range. The indicated proteins were incubated with recombinant active caspase-1 for 2 h at 37 degC. C , caspase-3-mediated processing of the purified recombinant proteins over the indicated concentration range. The indicated proteins were incubated with recombinant active caspase-3 for 2 h at 37 degC. Results are representative of at least three independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: In vitro and in situ interactions of Usp19, DNAAF 2, NADSYN 1, p23/ PTGES 3, Hsp105 and Bcl-G with dc TPR proteins. (A) Coomassie blue R staining of overexpressed and purified proteins used in GST pull-down assay. (B) Western blot analysis of GST- AIP and GST- FKBP 51 pull-down of Usp19, DNAAF 2, NADSYN 1, p23/ PTGES 3, Hsp105, Bcl-G and their truncated versions lacking five utmost C-terminal residues. (C) PLA of Usp19, DNAAF 2, NADSYN 1, p23/ PTGES 3, Hsp105 and Bcl-G interacting with AIP in HEK 293 cells. The asterisk indicates position of truncated p23 on the gel.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Identification of the co-chaperones p23 and Cdc37 as promoters of opioid anti-nociception in the brain. Male CD-1 mice used for all experiments, data reported as the mean +- SEM, with sample sizes of mice/group noted in each graph. (A-C) Ten nanomoles of Gedunin (p23, A ), 10 nmol of Celastrol (Cdc37, B ), or 0.1 nmol of KU177 (Aha1, C ) or Vehicle control injected icv concurrently with paw incision surgery with a 24 h recovery, followed by 3.2 mg/kg morphine sc . Experiments performed with two technical replicates for each drug. *,**,**** p < 0.05, 0.01, 0.0001 vs. same time point inhibitor treatment group by two-way ANOVA with Fisher's Least Significant Difference post hoc test. (D) p23, Cdc37, or Negative Control CRISPR-treated mice with icv delivery of constructs analyzed by IHC for target knockdown on day 10. Representative images shown from the PRN. Both targets (p23 - red, Cdc37 - green) have a similar staining pattern to Hsp90alpha, and both are clearly reduced by CRISPR treatment. (E) Data from (D) for all mice quantitated as described in the ""Materials and Methods"" section. All mice treated in one technical replicate, with staining and analysis of the resulting tissue performed in more than one technical replicate. * p < 0.05 vs. same target Negative Control group by Unpaired 2-Tailed t -test. CRISPR treatment reduced p23 signal by 36.3% and Cdc37 by 46.0%. (F) CRISPR-treated mice had paw incision surgery performed on day 9, with injection of 3.2 mg/kg mo

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 The annular ring of chaperones is a pre-assembled structure. (a) Undifferentiated N2a cells were stained with the Ac88 clone anti-hsp90 (red), which does not recognize the chaperone unless it is free or denatured. FKBP52 was used as marker of the perinuclear ring (green). (b) Indirect immunofluorescence for N2a cells pre-incubated for 30 min with cycloheximide (+CHX) followed by stimulation with FK506 for 3 h (+CHX + FK506) without changing the medium. (-CHX): Control of untreated cells. Bars = 10 mum.