Total proteins were extracted from epididymal tissues using Tissue Protein Extraction Reagent (Thermo Scientific) containing 1 mM of PMSF (Roche). The protein concentrations were determined by BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein samples were separated by SDS/PAGE (10% (w/v) gel) and transferred on to PVDF membranes (Millipore, Billerica, MA, U.S.A.). The transferred proteins were incubated with polyclonal rabbit anti-ZO-1 (1:500, Santa Cruz Biotechnology), polyclonal rabbit anti-vimentin (1:2000, CST, Danvers, MA, U.S.A.), and polyclonal rabbit anti-GAPDH (1:10000, Abcam) primary antibodies at 4°C overnight, followed by incubation with the horseradish peroxidase conjugated anti-rabbit (1:10000, KPL, Milford, MA, U.S.A.) secondary antibody for 1 h at 37°C. The reaction was developed with the ECL kit (Pierce Chemical, Dallas, TX, U.S.A.) and photographed.
Rabbit polyclonal anti gapdh
Manufactured by Abcam
Sourced in United States
Rabbit polyclonal anti‐GAPDH is a laboratory reagent used for the detection and quantification of GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Polyclonal rabbit anti zo 1, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions)
Goat anti mouse irdye 680, by LI COR (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti giantin, by Abcam (1 mentions)
Mouse monoclonal anti α tubulin, by Merck Group (1 mentions)
Mouse monoclonal anti ha tag, by Cell Signaling Technology (1 mentions)
Alexa fluor 568 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rabbit polyclonal anti ha, by Merck Group (1 mentions)
Mouse anti erp72, by Santa Cruz Biotechnology (1 mentions)
Goat anti sel1l, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg peroxidase, by Merck Group (1 mentions)
Rabbit anti mouse igg peroxidase, by Merck Group (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti human cd3, by BD (1 mentions)
Mouse monoclonal anti vinculin, by Abcam (1 mentions)
5 protocols using rabbit polyclonal anti gapdh
1
Western Blot Analysis of Epididymal Proteins
2
Monoclonal Antibodies for AAT Detection
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Monoclonal mouse anti-total antitrypsin 2G7 (1:100 v/v)61 (link) was used to detect both monomeric and polymer forms of AAT. Monoclonal mouse anti-polymer 2C1 antibody (1:25 v/v, culture media supernatant)62 (link) recognised the AAT polymer. Polyclonal rabbit anti-GAPDH (#ab9485, 1:5,000 v/v) and rabbit anti-Giantin (#ab24586, 1:100 v/v) antibodies were obtained from Abcam. Polyclonal rabbit anti-Calreticulin (PA3–900, 1:500 v/v) was obtained from Thermo Scientific. The secondary antibodies donkey anti-mouse Alexa Fluor 555 and donkey anti-rabbit Alexa Fluor 488 (1:1,000 v/v) used in immunocytochemistry were obtained from Molecular Probes, Invitrogen. Goat anti-mouse IRDye 680 and anti-rabbit IRDye 800 infrared secondary antibodies used for western blotting (1:20,000 v/v) were purchased from LI-COR Biosciences.
3
Immunofluorescence and Western Blotting Antibodies
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The antibodies with their dilutions and sources were as follows: antibodies for immunofluorescence: mouse monoclonal anti‐HA‐tag [1 : 200; Cell Signaling Technologies (CST; Danvers, MA, USA)], rabbit polyclonal anti‐calnexin (CANX, 1 : 200; Santa Cruz Biotechnology, Dallas, TX, USA), Alexa Fluor 568‐goat anti‐mouse IgG (1 : 200; Molecular Probes, Eugene, OR, USA), Alexa Fluor 647‐goat anti‐rabbit IgG (1 : 200; Molecular Probes). Antibodies for western blotting: rabbit polyclonal anti‐HA (1 : 4000; Sigma‐Aldrich, St. Louis, MO, USA), anti‐Histone H3(1 : 1000, CST), rabbit polyclonal anti‐GAPDH (1 : 2500; Abcam, Cambridge, UK), mouse monoclonal anti‐α‐tubulin (1 : 10 000; Sigma‐Aldrich), rabbit anti‐ CANX (1 : 1000; CST), rabbit anti‐BiP (1 : 1000, CST), rabbit anti‐GRP94 (1 : 1000, CST), mouse anti‐ERP72 (1 : 200; Santa Cruz Biotechnology), goat anti‐SEL1L (1 : 200; Santa Cruz Biotechnology), rabbit anti‐HRD1 (1 : 500; CST), rabbit anti‐OS‐9 (1 : 500; Abcam), goat anti‐rabbit IgG‐peroxidase (1 : 50 000; Sigma‐Aldrich), and rabbit anti‐mouse IgG‐peroxidase (1 : 80 000; Sigma‐Aldrich).
4
Reagents and Antibodies Used in Research
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Reagents, including LPS and rabbit anti-JMJD6 (PSR) antibody, were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Aralast NP and Prolastin C was from Baxter International Inc. (Chicago, IL, USA) and Grifols (LA, CA, USA), respectively. Phorbol 12-myristate 13-acetate (PMA), rabbit anti-MAR antibody, rabbit anti-iNOS antibody, rabbit polyclonal anti-GAPDH, mouse monoclonal anti-vinculin were from Abcam (Cambridge, MA, USA), rabbit anti-SRB-2 antibody was from Santa Cruz Biotechnology (Dallas, TX, USA), mouse anti-human CD3 was from BD Biosciences (Franklin Lakes, NJ, USA), recombinant rat IL-4 was from ProSpec-Tany TechnoGene (Ness-Ziona, Israel), and TNF-α Protease Inhibitor-1 (TAPI-1) was from ESD Millipore (Billerica, MA, USA).
5
Western Blot Analysis of Angiogenic and Mineralization Markers
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The protein extracts from the four groups of cells were separated on 12% sodium dodecyl sulfate-polyacrylamide gels (Beyotime Institute of Biotechnology, Haimen, China) and transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Inc.) at 200 mA for 2 h. The membranes were blocked with 5% non-fat milk for 2 h, and were incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal anti-VEGF (1:500; cat. no. ab46154; Abcam, Cambridge, MA, USA); rabbit polyclonal anti-BMP2 (1:500; cat. no. ab14933; Abcam) and rabbit polyclonal anti-dentin sialoprotein (DSP; 1:500; cat. no. sc-33586; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were subsequently incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:20,000; cat. no. ab97051; Abcam) at 37°C for 2 h. Rabbit polyclonal anti-GAPDH (1:2,500; cat. no. ab9485; Abcam) was selected as the internal control. The resultant bands were visualized by the Chemiluminescence Western Blotting Detection system (EMD Millipore) on X-ray films (Kodak, Rochester, NY, USA).
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