MA3-018

antibody from Invitrogen Antibodies

Targeting: P4HB DSI, ERBA2L, GIT, P4Hbeta, PDI, PDIA1, PO4DB, PO4HB, PROHB

Provider product page for MA3-018

Western blot

Antibody data

Antibody Data
Antigen structure
References [44]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [6]
Immunohistochemistry [3]
Flow cytometry [3]
Other assay [9]

Submit

Product number: MA3-018 - Provider product page
Provider: Invitrogen Antibodies
Product name: PDI Monoclonal Antibody (RL77)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-018 detects protein disulphide-isomerase (PDI) from human, mouse, rat, porcine, xenopus liver tissues as well as hamster (CHO) cells and canine samples.
Antibody clone number: RL77
Concentration: Conc. Not Determined

P4HB protein structure - MA3-018 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of PDI was performed by loading 25 µg of HepG2 (Lane 1), Hela (Lane 2) and NIH-3T3 (Lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a PDI monoclonal antibody (Product # MA3-018) 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 approx. 57 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of PDI was achieved by transfecting A-431 cells with PDI specific siRNAs (Silencer® select Product # s441). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the PDI knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-PDI Mouse Monoclonal Antibody (Product # MA3-018, 1:2000 dilution) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). 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 PDI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-PDI Mouse Monoclonal Antibody (Product # MA3-018) and a 57 kDa band corresponding to PDI was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2),NIH/3T3 (Lane 3), A-375 (Lane 4), Hep G2 (Lane 5), HL-60 (Lane 6), and PC-12 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 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)
Main image
Experimental details: Immunofluorescent analysis of PDI (green) in MDCK cells. The cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 15 minutes at room temperature. Cells were stained with a PDI mouse monoclonal antibody (Product # MA3-018), at a dilution of 1:100 in blocking buffer for at least 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate (Product # 35502) at a dilution of 1:1000 for 30 minutes at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PDI using anti-PDI monoclonal antibody (Product # MA3-018) shows staining in A549 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PDI using anti-PDI monoclonal antibody (Product # MA3-018) shows staining in HMVEC Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PDI using anti-PDI monoclonal antibody (Product # MA3-018) shows staining in NS-1 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunolocalization of PDI in rat epithelial cells using Product # MA3-018.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunolocalization of PDI in rat epithelial cells using Product # MA3-018.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human colon carcinoma tissues. 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 1:20 with a mouse monoclonal antibody recognizing PDI (Product # MA3-018) 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 deparaffinized Human liver tissue tissues. 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 1:200 with a mouse monoclonal antibody recognizing PDI (Product # MA3-018) 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 deparaffinized Human tonsil tissue tissues. 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 1:200 with a mouse monoclonal antibody recognizing PDI (Product # MA3-018) 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: Flow cytometry analysis of PDI 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 PDI monoclonal antibody (Product # MA3-018) 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 PDI in K562 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 PDI monoclonal antibody (Product # MA3-018) 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 PDI 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 PDI monoclonal antibody (Product # MA3-018) 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: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. ER-localized USP20 and USP33 are required for deubiquitination of TA proteins . (A) HEK293 cells with the indicated CRISPR/Cas9 knockout cell lines were transfected with VAPA-FLAG and HA-ubiquitin. VAPA was immunoprecipitated (IP) and immunoblotted (IB) with anti-FLAG antibody for VAPA and anti-HA antibody for ubiquitinated (Ub) VAPA. The orange circle indicates the glycosylated form of VAPA. (B) WT HEK293 and USP20/33 -/- cells were fractionated into cytosol (cyto.) and membrane (mem.) using 0.015% digitonin. Immunoblotting of TRAPalpha (membrane control) and SGTA (cytosolic control) shows the successful separation of the cytosol and membrane. Data are representative of two independent experiments. (C) Increasing amounts of rough microsomes isolated from HEK293 cells were immunoblotted for USP20, USP33, and the membrane protein Sec61alpha as a control. (D) WT, USP20 -/- , and USP33 -/- HEK293 cells were stained for the indicated endogenous proteins. Confocal imaging shows colocalization signal (yellow) of USP33 (red) and calreticulin (green). Scale bar, 10 um. Data are representative of two independent experiments. (E) HeLa cells were transfected with the ER control protein PDI and either USP20 or USP33 and then treated as in C for confocal microscopy. Scale bar, 10 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure S2. Endogenous USP20 localization. Either WT or USP20 -/- HEK293 cells were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton X-100. The cells were stained with rabbit alpha-USP20 and mouse alpha-PDI followed by goat alpha-rabbit IgG-Cy2 and goat alpha-mouse IgG-Cy3. The stained cells were examined for colocalization (yellow) of USP20 (red) and PDI (green) by confocal microscopy. Scale bar, 10 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 GBF1 Depletion Causes an Accumulation of Unprocessed VWF in Endothelial Cells (A) Amount of VWF mRNA (relative to mock cells). n = 5 independent experiments, SEM, unpaired t test, p = 0.002. (B) Total amount of VWF protein measured by western blot (WB) (relative to mock cells). n = 4 independent experiments, SEM, unpaired t test, n.s. = not significant. (C) Representative western blot showing GBF1 protein depletion and VWF processing. ""pro-VWF,"" processed VWF and ""unp-VWF,"" unprocessed VWF. (D) Percentage of pro-VWF from total VWF (quantification from WB). n = 4 independent experiments, SEM, unpaired t test, p < 0.0001. (E) Immunofluorescence images of HUVECs stained with antibodies targeting unp-VWF, green and pro-VWF, red. Scale bars, 10 mum. (F) Immunofluorescence images of HUVECs stained for the ER-transmembrane protein calnexin, green and unp-VWF, red. Scale bars, 10 mum. (G) Immunofluorescence images of HUVECs stained for the ER-luminal protein calreticulin, green and unp-VWF, red. Scale bars, 10 mum. (H) Immunofluorescence images of HUVECs stained for the ER-luminal protein PDI, green and unp-VWF, red. Scale bars, 10 mum. See also Figure S3 .