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

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

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

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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. MA3-018 has been successfully used in Western blot, immunofluorescence, immunohistochemical, flow cytometry, and immunoprecipitation procedures. By Western blot, this antibody detects a 59 kDa protein representing PDI from rat liver extract or a slightly higher protein at 61 kDa representing PDI from human liver extract. Immunohistochemical staining of PDI in rat intestine with MA3-018 yields a pattern consistent with cytoplasmic staining. In immunoprecipitation procedures MA3-018 has been shown to inhibit the activity of PDI in vitro. In immunohistochemistry procedures, formalin-fixed paraffin-embedded tissue sections are recommended. The MA3-018 antigen is purified rat PDI.
Reactivity: Human, Mouse, Rat, Canine, Hamster, Porcine, Xenopus
Host: Mouse
Isotype: IgG
Antibody clone number: RL77
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

P4HB protein structure - MA3-018 shown in red.

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)
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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)
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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)
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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)
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Experimental details: Immunolocalization of PDI in rat epithelial cells using Product # MA3-018.

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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)
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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)
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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)
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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: 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)
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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)
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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 .