Anti-ENPP1/PC1 antibody [EPR22262-22]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (human liver hepatocellular carcinoma cell line) cells labeling ENPP1/PC1 (green) with ab223268 at 1/100 dilution, followed by Alexa Fluor^® 488 Goat anti-Rabbit secondary antibody (ab150077) at 1/1000 dilution. Confocal image showing membranous staining in HepG2 cells. Tubulin was detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red). The nuclear countertsain was DAPI (Blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Alexa Fluor^® 488 Goat anti-Rabbit (ab150077) at 1/200 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HepG2 (human liver hepatocellular carcinoma cell line) cells labeling ENPP1/PC1 with ab223268 (red) compared with a Rabbit monoclonal IgG isotype control (ab172730) (black)and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

Immunohistochemical analysis of paraffin-embedded human pancreas tissue stained for ENPP1/PC1 using ab223268 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human pancreas (PMID : 9344668; PMID : 23861746) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MDA-MB-231 (human breast adenocarcinoma cell line) cells labeling ENPP1/PC1 (green) with ab223268 at 1/100 dilution, followed by Alexa Fluor^® 488 Goat anti-Rabbit secondary antibody (ab150077) at 1/1000 dilution. Confocal image showing membranous staining in MDA-MB-231 cells. Tubulin was detected using Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (Red). The nuclear countertsain was DAPI (Blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Alexa Fluor^® 488 Goat anti-Rabbit (ab150077) at 1/200 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

Immunohistochemical analysis of paraffin-embedded human liver tissue stained for ENPP1/PC1 using ab223268 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human liver (PMID : 25539584; PMID : 9344668; PMID : 23861746) is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

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Immunoprecipitation - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

ENPP1/PC1 was immunoprecipitated from 0.35 mg of MDA-MB-231 (human breast adenocarcinoma cell line) whole cell lysate using ab223268 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab223268 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1 : MDA-MB-231 whole cell lysate 10μg (input)
Lane 2 : ab223268 IP in MDA-MB-231 whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab223268 in MDA-MB-231 whole cell lysate (-).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-ENPP1/PC1 antibody [EPR22262-22] (ab223268)

Predicted band size: 105 kDa

Observed band size: 130 kDa

false

• WB

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Western blot - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

Blocking and dilution buffer : 5% NFDM/TBST

Exposure time :

Lane 1 : 3 minutes;
Lane 2 : 70 seconds;
Lane 3 : 15 seconds;
Lane 4 : 3 minutes

The molecular weight observed is consistent with what has been described in the literature (PMID : 19577557).

All lanes:

Western blot - Anti-ENPP1/PC1 antibody [EPR22262-22] (ab223268) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

MDA-MB-231 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 3:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Huh7 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lanes 2 - 4:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 105 kDa

Observed band size: 130 kDa

false

• WB

CiteAb

Western blot - Anti-ENPP1/PC1 antibody [EPR22262-22] (AB223268)

ENPP1/PC1 western blot using anti-ENPP1/PC1 antibody [EPR22262-22] ab223268. Publication image and figure legend from Luo, X., Cui, H., et al., 2020, Front Immunol, PubMed 32425926.

ab223268 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab223268 please see the product overview.

AFP TCR 2 is unlikely to cross-react with other proteins in the human genome. (A) Graphic presentation of X-scan results showing the replaceability of each amino acid of AFP158−166 epitope. Peptides with replacement of each amino acid residue with every other 19 amino acids were used to activate the TCR 2 transduced T cells. The amount of IFN-γ produced by AFP TCR 2 transduced T cells after stimulation with mutated epitope (mut) was compared to that from wild type AFP158−166 peptide (wt) and the ratios are presented. Dashed line indicates the cut-off for tolerance. Data represents the mean + s.d. of duplicate co-cultures and is representative of 3 healthy donors. (B) ENPP1 and AFP expression in the indicated cell lines tested by Western blot. GAPDH was used as loading control. (C) IFN-γ concentrations in the supernatant of overnight, 1 : 1 ratio co-culture of UT or AFP TCR 2 transduced T cells with the indicated cell lines measured by ELISA. Data represents the mean + s.d. of quadruplicate co-cultures and is representative of 3 healthy donors.

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