Human Protein Disulfide Isomerase/P4HB Antibody (537331)

R&D Systems | Catalog # MAB4236

R&D Systems

Key Product Details

Species Reactivity

Human

Applications

Western Blot, Immunocytochemistry, Simple Western, Immunoprecipitation

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG2A Clone # 537331
View all Protein Disulfide Isomerase/P4HB Primary Antibodies »

Product Specifications

Immunogen

Chinese hamster ovary cell line CHO-derived recombinant human Protein Disulfide Isomerase/P4HB
Asp18-Lys505
Accession # P07237

Specificity

Detects human Protein Disulfide Isomerase/P4HB in direct ELISAs and Western blots.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG2A

Scientific Data Images for Human Protein Disulfide Isomerase/P4HB Antibody (537331)

Detection of Human Protein Disulfide Isomerase/P4HB antibody by Western Blot.

Detection of Human Protein Disulfide Isomerase/P4HB by Western Blot.

Western blot shows lysates of PANC-1 human pancreatic carcinoma cell line and MCF-7 human breast cancer cell line. PVDF membrane was probed with 1 µg/mL of Mouse Anti-Human Protein Disulfide Isomerase/P4HB Monoclonal Antibody (Catalog # MAB4236) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for Protein Disulfide Isomerase/P4HB at approximately 57 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Protein Disulfide Isomerase/P4HB antibody in HeLa Human Cell Line by Immunocytochemistry (ICC).

Protein Disulfide Isomerase/P4HB in HeLa Human Cell Line.

Protein Disulfide Isomerase/P4HB was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Mouse Anti-Human Protein Disulfide Isomerase/P4HB Monoclonal Antibody (Catalog # MAB4236) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the Northern-Lights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
Detection of Human Protein Disulfide Isomerase/P4HB antibody by Western Blot.

Detection of Human Protein Disulfide Isomerase/P4HB by Western Blot.

Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line and human peripheral blood mononuclear cells (PBMC). PVDF Membrane was probed with 2 µg/mL of Human Protein Disulfide Isomerase/P4HB Monoclonal Antibody (Catalog # MAB4236) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for Protein Disulfide Isomerase/P4HB at approximately 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Protein Disulfide Isomerase/P4HB by Simple WesternTM.

Simple Western lane view shows lysates of PANC‑1 human pancreatic carcinoma cell line, loaded at 0.2 mg/ml. A specific band was detected for Protein Disulfide Isomerase/P4HB at approximately 59 kDa (as indicated) using 10 µg/mL of Mouse Anti-Human Protein Disulfide Isomerase/P4HB Monoclonal Antibody (Catalog # MAB4236) followed by 1:50 dilution of HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). This experiment was conducted under reducing conditions and using the 12-230kDa kDa separation system.
Detection of P4HB by Western Blot

Detection of P4HB by Western Blot

Knockdown of PDIA1 in Hep-TR alpha and Hep-TR beta cell lines followed by qPCR. (A) Knockdown of PDI in Hep-TRs cell lines in T3 presence (+T3). Western blot shows the absence of PDIA1 protein in knockdown samples (PDIA1 siRNA). Cells were treated with 1 μM of T3 for 6 h prior to cell harvesting. We presented here only 2 out of 3 knockdown experiments for each condition. Antibodies: anti-PDI and anti-beta -Actin as loading control. (B) Knockdown of PDI in Hep-TRs cell lines in T3 absence (–T3). Western blot shows the absence of PDIA1 protein in knockdown samples (PDIA1 siRNA). Cells were treated with 1 μM of DMSO for 6 h prior to cell harvesting. We presented here only 2 out of 3 knockdown experiments for each condition. Antibodies: anti-PDI and anti-beta -Actin as loading control. (C) qPCRs for each cell line, Hep-TR alpha and Hep-TR beta, with or without T3. Hif2a and Myh6 in absence of PDI (knockdown condition), increases transcription, Furin in absence of PDI (knockdown condition), did not alter gene expression. Each experiment was performed in three biological replicates. Statistical Analysis with One-way ANOVA *p < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30671024), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of P4HB by Western Blot

Detection of P4HB by Western Blot

Co-immunoprecipitation of TR:PDIA1 in 293T cells shows the interaction for both isoforms with or without T3 presence. (A) Immunoprecipitation of PDIA1 followed by Western Blot anti-flag, revealing Co-IP of PDIA1 and flag-TRs. The treatment with T3 (1 μM) decreases TR expression in 293T cell (input bands are thinner) reducing protein available to be immunoprecipitated. The interactions of TRs with PDIA1 were confirmed according to the quantification of bands (presented in the graphs below the WB). (B) Immunoprecipitation of flag tagged TRs (+ or –T3), and western blot anti-PDIA1. Control experiment WB anti-flag is presented in Supplementary Figure 2A, together with a confirmation experiment (Supplementary Figure 2B). Although WB shows strong IgG band, it is still possible to observe the presence of PDIA1 in all conditions, co-immunoprecipitated with both isoforms of TR. The quantification of bands are presented in graphs below WB, where all signals were normalized to the control signal (relative Density). 293T cell extracts without T3 and without transfection of flag-TRs were used on this experiment as negative control. Co-IP and WB experiments were performed more than once and here we show a representative image. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30671024), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of P4HB by Western Blot

Detection of P4HB by Western Blot

Co-immunoprecipitation of TR:PDIA1 in 293T cells shows the interaction for both isoforms with or without T3 presence. (A) Immunoprecipitation of PDIA1 followed by Western Blot anti-flag, revealing Co-IP of PDIA1 and flag-TRs. The treatment with T3 (1 μM) decreases TR expression in 293T cell (input bands are thinner) reducing protein available to be immunoprecipitated. The interactions of TRs with PDIA1 were confirmed according to the quantification of bands (presented in the graphs below the WB). (B) Immunoprecipitation of flag tagged TRs (+ or –T3), and western blot anti-PDIA1. Control experiment WB anti-flag is presented in Supplementary Figure 2A, together with a confirmation experiment (Supplementary Figure 2B). Although WB shows strong IgG band, it is still possible to observe the presence of PDIA1 in all conditions, co-immunoprecipitated with both isoforms of TR. The quantification of bands are presented in graphs below WB, where all signals were normalized to the control signal (relative Density). 293T cell extracts without T3 and without transfection of flag-TRs were used on this experiment as negative control. Co-IP and WB experiments were performed more than once and here we show a representative image. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30671024), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of P4HB by Western Blot

Detection of P4HB by Western Blot

Knockdown of PDIA1 in Hep-TR alpha and Hep-TR beta cell lines followed by qPCR. (A) Knockdown of PDI in Hep-TRs cell lines in T3 presence (+T3). Western blot shows the absence of PDIA1 protein in knockdown samples (PDIA1 siRNA). Cells were treated with 1 μM of T3 for 6 h prior to cell harvesting. We presented here only 2 out of 3 knockdown experiments for each condition. Antibodies: anti-PDI and anti-beta -Actin as loading control. (B) Knockdown of PDI in Hep-TRs cell lines in T3 absence (–T3). Western blot shows the absence of PDIA1 protein in knockdown samples (PDIA1 siRNA). Cells were treated with 1 μM of DMSO for 6 h prior to cell harvesting. We presented here only 2 out of 3 knockdown experiments for each condition. Antibodies: anti-PDI and anti-beta -Actin as loading control. (C) qPCRs for each cell line, Hep-TR alpha and Hep-TR beta, with or without T3. Hif2a and Myh6 in absence of PDI (knockdown condition), increases transcription, Furin in absence of PDI (knockdown condition), did not alter gene expression. Each experiment was performed in three biological replicates. Statistical Analysis with One-way ANOVA *p < 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30671024), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human Protein Disulfide Isomerase/P4HB Antibody (537331)

Application
Recommended Usage

Immunocytochemistry

8-25 µg/mL
Sample: Immersion fixed HeLa human cervical epithelial carcinoma cell line

Immunoprecipitation

25 µg/mL
Sample: Conditioned cell culture medium spiked with Recombinant Human P4HB (Catalog # 4236-DI), see our available Western blot detection antibodies

Simple Western

10 µg/mL
Sample: PANC-1 human pancreatic carcinoma cell line

Western Blot

1-2 µg/mL
Sample: PANC‑1 human pancreatic carcinoma cell line, MCF‑7 human breast cancer cell line, HeLa human cervical epithelial carcinoma cell line and human peripheral blood mononuclear cells (PBMC)

Formulation, Preparation, and Storage

Purification

Protein A or G purified from hybridoma culture supernatant

Reconstitution

Sterile PBS to a final concentration of 0.5 mg/mL. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Protein Disulfide Isomerase/P4HB

P4HB (Prolyl 4-hydroxylase beta chain; also PDI) is a 60 kDa member of the protein disulfide isomerase family.  As an intracellular homodimer, it forms a tetrameric complex with P4H alpha chains to form an active prolyl 4-hydrolyase.  This catalyses the hydroxylation of proline in collagen.  On the cell surface, it reduces disulfide bonds in HIV that allow the virus to fuse with CXCR4 and enter susceptible cells.  Mature human P4HB is 491 amino acids (aa) in length.  It contains two TRX domains (aa 25-134 and 368-475) plus an ER retention sequence (aa 505-508).  There is one potential isoform that shows an 11 aa substitution for the first 162 amino acids. Over aa 18-505, human P4HB shares 94% aa identity with mouse P4HB. 

Long Name

Proline 4-Hydroxylase, Beta Polypeptide

Alternate Names

DSI, ERBA2L, GIT, PDI, PDIA1, PO4DB, PO4HB, PROHB, Thbp

Entrez Gene IDs

5034 (Human); 18453 (Mouse); 25506 (Rat)

Gene Symbol

P4HB

UniProt

P07237

Additional Protein Disulfide Isomerase/P4HB Products

Product Documents for Human Protein Disulfide Isomerase/P4HB Antibody (537331)

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Product Specific Notices for Human Protein Disulfide Isomerase/P4HB Antibody (537331)

For research use only

Related Research Areas

Redox Enzymes

Citations for Human Protein Disulfide Isomerase/P4HB Antibody (537331)

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