Native and polymeric forms of AAT were detected in HepG2 cells transfected with M and Z alleles as previously described [17 (link)]. Quantification of the Western blot bands of the AAT protein in MM-ORG and ZZ-ORG was performed with Image J software v1.48. The antibodies used for Western blotting were: rabbit polyclonal antisera against human AAT (1:1000; Dako, Denmark); antibody D11 (1:800) [54 (link)] for the detection of AAT aggregates; and monoclonal antibody AC-74 anti-beta-actin (1:5000; Sigma Aldrich). The secondary antibodies used were chicken anti-mouse IgG-HRP (sc-2954 Santa Cruz Biotechnology, USA) and donkey anti-rabbit (Cytiva Europe GMBH, Barcelona Spain), both at 1:5000 dilution.
Chicken anti mouse igg hrp sc 2954
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Chicken anti-mouse IgG-HRP (sc-2954) is a secondary antibody conjugate that binds to mouse immunoglobulin G (IgG). The antibody is labeled with horseradish peroxidase (HRP), which allows for detection and visualization in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Immobilon hrp substrate, by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ab34710, by Abcam (1 mentions)
Sc 2357 cm, by Santa Cruz Biotechnology (1 mentions)
Mouse anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Signalfire ecl reagent, by Cell Signaling Technology (1 mentions)
Mab374, by Merck Group (1 mentions)
Ab133303, by Abcam (1 mentions)
α tubulin t5168, by Merck Group (1 mentions)
Donkey anti goat igg hrp sc 2020, by Santa Cruz Biotechnology (1 mentions)
Immun star hrp chemiluminescent kit, by Bio-Rad (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
4 protocols using chicken anti mouse igg hrp sc 2954
1
Quantification of AAT Protein Expression
2
Characterization of AAT Protein Variants in Organoids
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Native and polymeric forms of AAT protein were detected in cell pellets and culture medium of expanding and differentiated organoids. Cell pellets were lysed with RIPA buffer containing a cocktail of complete protease inhibitors (Roche, Mannhein, Germany). The lysates and culture media were electrophoresed on 10% polyacrylamide sodium dodecyl sulfate PAGE (SDS-PAGE). The insoluble fraction was sonicated and separated on non-denaturing 8% PAGE. Proteins were detected with the following primary antibodies: anti-AAT B9 (1:100 dilution) and anti-beta-actin (AC-74 Sigma Aldrich, Taufkirchen, Germany) (1:5000 dilution) followed by chicken anti-mouse IgG-HRP (sc-2954 Santa Cruz Biotechnology) (1:5000 dilution), and visualized after labeling with Immobilon HRP substrate (Millipore) using Chemidoc Touch imaging system (Biorad, Hercules, CA, USA). Quantification of the western blot bands of AAT protein of MM, MZ and ZZ organoids was performed with Image J software.
3
Baicalin/Baicalein Modulate Collagen I Expression
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After a 48 h treatment with baicalin/baicalein (20–80 µM), total protein was extracted from NRK-49F cell lysate and the protein concentration was determined using the bicinchoninic acid assay. Protein samples (20 µg) were loaded and separated on a 10% gel using SDS-PAGE, and then electrotransferred to nitrocellulose membranes. After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with rabbit anti-rat polyclonal COL1 primary (1:1,000, ab34710; Abcam) and mouse anti-rat monoclonal GAPDH (1:1,000, MAB374; Merck KGaA) primary antibodies overnight at 4°C, followed by incubation with a secondary antibody (mouse anti-rabbit IgG-HRP, sc-2357-CM, and chicken anti-mouse IgG-HRP (sc-2954; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 1:5,000 dilutions for 1 h at room temperature. Protein bands were then detected using SignalFire ECL Reagent (Cell Signaling Technology, Inc., Danvers, MA, USA).
4
Mitochondrial Protein Quantification and Analysis
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Protein concentration in the isolated mitochondria from the nigral region (n=4) and whole homogenate (n=4) were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Fremont, CA, USA). Equal amounts of protein were separated on a 5–10% bis-tris polyacrylamide gel, and transferred to nitrocellulose membranes. Membranes were incubated overnight with primary antibodies against VDAC/porin (V2139, 1 : 1000) and α-Tubulin (T5168, 1 : 50 000) from Sigma, HDAC2 (sc-56685, 1 : 200), AT1 (sc-31181, 1 : 200) and AT2 (sc-9040, 1 : 200) from Santa Cruz Biotechnology and rabbit monoclonal antibody to Nox4 (ab133303, 1 : 800) from Abcam. The following HRP-conjugated secondary antibodies were used: Protein A (1 : 5000) (NA9120V; GE Healthcare), chicken anti-mouse IgG-HRP (sc-2954, 1 : 2500) and donkey anti-goat IgG-HRP (sc-2020, 1 : 2500) from Santa Cruz Biotechnology. Immunoreactive bands were detected with an Immun-Star HRP Chemiluminescent Kit (170-5044; Bio Rad) and visualized with a chemiluminescence detection system (Molecular Imager ChemiDoc XRS System; Bio Rad). Specificity of the antibodies was confirmed as indicated above.
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