Anti-alpha 1 Antitrypsin antibody [EPR17087-50]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling alpha 1 Antitrypsin with ab207303 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on cancer cells of human hepatocellular carcinoma 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) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling alpha 1 Antitrypsin with ab207303 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on human liver 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) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling alpha 1 Antitrypsin with ab207303 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on K562 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse mAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab207303 at 1/50 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse mAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma) cells labeling alpha 1 Antitrypsin with ab207303 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse mAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse Alexa Fluor^® 594 (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab207303 at 1/50 dilution, followed by Goat Anti-Mouse Alexa Fluor^® 594 (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse mAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) seconday antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

alpha 1 Antitrypsin was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell lysate with ab207303 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab207303 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1 : HepG2 whole cell lysate 10ug (Input).
Lane 2 : ab207303 IP in HepG2 whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207303 in HepG2 whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (ab207303)

Predicted band size: 46 kDa

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• WB

Collaborator

Western blot - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

ab207303 was shown to react with SERPINA1 in wild-type HepG2 cells in Western blot with loss of signal observed in a SERPINA1 knockout cell line. Wild-type HepG2 and SERPINA1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab207303 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (ab207303) at 1/5000 dilution

Lane 1:

Wild-type HepG2 lysate at 30 µg

Lane 2:

SERPINA1 knock-out HepG2 lysate at 30 µg

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• WB

Supplier Data

Western blot - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (ab207303) at 1/5000 dilution

All lanes:

Human fetal liver lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG. at 1/10000 dilution

Predicted band size: 46 kDa

Observed band size: 51 kDa,55 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (ab207303) at 1/5000 dilution

Lane 1:

Human liver lysate at 20 µg

Lane 2:

Human heart lysate at 20 µg

Lane 3:

Human kidney lysate at 20 µg

Lane 4:

Human spleen lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 46 kDa

Observed band size: 51 kDa,55 kDa

false

Exposure time: 1min

• WB

CiteAb

Western blot - Anti-alpha 1 Antitrypsin antibody [EPR17087-50] (AB207303)

alpha 1 Antitrypsin western blot using anti-alpha 1 Antitrypsin antibody [EPR17087-50] ab207303. Publication image and figure legend from Sarabhai, T., Peter, C., et al., 2017, PLoS One, PubMed 28493974.

ab207303 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 ab207303 please see the product overview.

Serum from trauma patients induces intrinsic apoptosis resistance by up-regulating AAT expression in neutrophils.A. Levels of AAT were quantified in the serum isolated from healthy volunteers (n = 6) and major trauma patients (n = 13) at days 1–2, days 5–6 and days 10–11 after admission by Elisa. *p<0.05; **p<0.01 (Kruskal-Wallis test with Dunns post hoc test) B. Additionally, AAT gene expression levels were quantified in neutrophils isolated from healthy volunteers and trauma patients by Real time PCR. Gene expression was normalized to that of 18S RNA. Expression levels are reflected as fold-change of expression relative to control levels (healthy volunteers). No significant differences were found. C. Neutrophils from healthy donors (2.5 × 106/ml) were cultured in medium containing FCS (1%) or pooled patient serum (1%) and treated with STS (0.2 μM). After 14 h, 3 μg/ml Brefeldin A was added to the cultures and cells were further cultured for additional 4 h. AAT gene expression was quantified by Real time PCR. Values were normalized to 18S RNA expression. Results of seven independent experiments are depicted. No statistical differences could be detected (one-way ANOVA with Newman keuls post-hoc test). D. AAT protein expression was analyzed in neutrophils after 18 h of culture by western blot analysis. Relative expression was quantified vs. GAPDH expression. One representative blot of five independent experiments is depicted. **p<0.01 (one-way ANOVA with Newman keuls post-hoc test). E. Neutrophils from healthy donors (2.5 × 106/ml) were cultured in medium containing FCS (1%) or pooled patient serum (1%) overnight. AAT levels in culture supernatants were quantified by Elisa at 0 h as well as after 18 h. Results of three independent experiments are depicted. ***p<0.001; n.s. = not significant (one-way ANOVA with Newman keuls post-hoc test).

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