Western blots were carried out using the following antibodies at the indicated dilutions: αTcf7l1 (Santa Cruz sc8635, 1:500), αcMyc (Santa Cruz sc42, 1:500), αp‐cMycSer62 (Abcam ab185656, 1:1,000), αp‐cMycThr58 (Santa Cruz sc135647, 1:500), alpha‐tubulin (Abcam ab126165, 1:1,000), αgoat HRP (Abcam ab6741, 1:2,000), αmouse HRP (Abcam ab6728, 1:2,000), and αrabbit HRP (Abcam ab16284, 1:2,000).
Ab6741
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab6741 is a monoclonal antibody that recognizes the protein GAPDH. It can be used for the detection of GAPDH in various applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (8 mentions)
Ab8227, by Abcam (4 mentions)
Ab6728, by Abcam (3 mentions)
Ab6789, by Abcam (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ab126165, by Abcam (1 mentions)
Ab16284, by Abcam (1 mentions)
Macs separation column, by Miltenyi Biotec (1 mentions)
A5316, by Merck Group (1 mentions)
Ab97046, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Sc 372, by Santa Cruz Biotechnology (1 mentions)
Sc 79305, by Santa Cruz Biotechnology (1 mentions)
Macs protein a g microbeads, by Miltenyi Biotec (1 mentions)
Ab124716, by Abcam (1 mentions)
Apoptag red in situ apoptosis detection kit, by Merck Group (1 mentions)
Potassium chloride (kcl), by Carlo Erba (1 mentions)
Ferric citrate, by Merck Group (1 mentions)
Mgso4, by Carlo Erba (1 mentions)
30 protocols using ab6741
1
Antibody-Based Western Blot Analysis
2
Protein Extraction and Immunoblotting for USP21 in Cell Lysates
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Cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich; EMD Millipore) as previously described (22 (link)–24 (link)). Cells were centrifuged at 4°C for 10 min at 16,000 × g. Protein concentrations were determined by the Bradford assay (25 (link)). Aliquots containing 20 µg of total protein were separated by 10% sodium dodecyl-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (1:5,000, mouse monoclonal; A5316; Sigma-Aldrich; EMD Millipore). Appropriate secondary antibodies (1:3,000; rabbit anti-goat; ab6741; 1:5,000; rabbit anti-mouse; ab97046; Abcam, Cambridge, MA, USA) conjugated to horseradish peroxidase and enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK) were used to detect the bound primary antibodies. Co-IP was performed with cell lysate (500 µg) incubated with USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc.), relA (1:1,000; rabbit polyclonal; sc-372; Santa Cruz Biotechnology, Inc.) or non-specific-IgG antibodies (1:1,000; rabbit IgG, monoclonal; ab172730; Abcam) using µMACS™ Protein A/G MicroBeads and MACS® Separation Columns according to the manufacturer's protocol (Miltenyi Biotec, Auburn, USA).
3
Quantitative Analysis of Secretory and Dimeric IgA
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Primary antibodies were used as for anti-IgA monoclonal antibody (ab124716 and ab97181; Abcam, Cambridge, UK) followed by secondary anti-IgG H&L horseradish peroxidase (HRP) (ab6721 and ab6741; Abcam). The immunohistochemical staining was completed by automatic immunohistochemical instrument. The images were collected and imported into Image-Pro Plus software for quantitative analysis of IgA protein, then the average optical density of secretory IgA (sIgA) and dimeric IgA (dIgA) protein was calculated and recorded. The IgA expressed in intra-luminal is defined as sIgA, while dIgA is expressed in sub-epithelial.
4
Molecular Mechanism of Axl Signaling
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DMEM (high glucose, [4.5 g/L]), NaCl, FBS, and a BCA protein assay kit (Pierce, Rockford, IL, USA) were purchased from Euroclone (Milan, Italy); Na3PO4, MgSO4, NaH2PO4, KH2PO4, and KCl were purchased from Carlo Erba (Milan, Italy); ferric citrate, Trypsin-EDTA-Solution (T4174) and Collagenasi type IA (C9891) were purchased from Sigma (St. Louis, MO, USA); the primary antibody for Axl (sc-1097) and GAS6 (N-20 sc-1936) were purchased from Santa Cruz (Heidelberg, Germany); the primary antibody for LC3-II was purchased from Cell Signalling (Danvers, MA, USA), and the anti-rabbit secondary antibody was purchased from GeneTex (Irvine, CA, USA); the anti-goat secondary antibody (ab6741) was purchased from Abcam (Cambrige, UK); Hepes Buffer Solution, PVDF membrane Invitrogen/Applied Biosystem (Milan, Italy); PE Annexin V Apoptosis Detection Kit I (559763) was purchased from Bioscience. An ApopTag Red In Situ Apoptosis Detection kit S7165 was purchased from Chemicon. All other reagents were obtained from Sigma (St. Louis, MO, USA).
5
Western Blot Analysis of Membrane Proteins
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Membrane proteins were transferred from BN-PAGE to PVDF membranes. After blocking of the membranes for 1 h with 10% non-fat dry milk in 0.1% TBST (100 mM Tris–HCL, 150 mM NaCl, pH 7.5, 0.1% Tween 20), mouse anti-GABAB1 (Abcam, ab55051, Cambridge, UK; 1/5000). Goat anti-GABAB1a (sc-73401/2500), goat anti-MuscarinicM2 (Abcam, ab140473, Cambridge, UK; 1/3000) and rabbit anti-GABAB2 (Abcam, ab75838, Cambridge, UK; 1/5000). The antibodies were detected using horseradish peroxidase-conjugated secondary anti-rabbit IgG antibodies (Abcam, ab6721, Cambridge, UK; 1/10,000), anti-Goat IgG antibodies (Abcam, ab6741, Cambridge, UK; 1/5000), Anti-Mouse IgG antibodies (Abcam, 97035, Cambridge, UK; 1/10,000) Membranes were then developed with the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). All membranes were stained with Coomassie blue R-350 (GE Healthcare, Buckinghamshire, UK, Falsafi et al., 2012 (link)). Arbitrary optical densities of immunoreactive bands were measured by the Image J software program (http://rsb.info.nih.gov/ij/ ). Loading controls for the BN-PAGE western blot were carried out according to Welinder and Ekblad (Welinder and Ekblad, 2011 (link)).
6
Platelet Protein Analysis via SDS-PAGE
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Following aggregations, platelets were lysed by adding 62.5 μL of 4X SDS in 250 μL of washed platelets, and heated at 95°C for 5 minutes. Samples were stored at −20°C until further analysis. Proteins from the 3 mouse groups’ platelets were separated by 10% SDS‐PAGE followed by its transfer to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk for 1 hour at room temperature. Antibodies against CLEC‐2 (R&D Systems, AF1718) and pAkt (Cell Signalling, 9275) were incubated with the membranes overnight at 4°C, and a horseradish peroxidase–conjugated secondary antibody (Abcam, AB6741) was used for detection using the Western Lightning Ultra chemiluminescence kit (PerkinElmer).
7
Immunodetection of IL-33 and Associated Proteins
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The following reagents were used in this study: mouse monoclonal antibody for IL-33 (ALX-804-840-C100, Enzo Life Sciences Inc., Farmingdale, New York, USA), rabbit polyclonal antibody for IL-33 (12372-1-AP, Proteintech Group Inc., Chicago, USA), goat polyclonal antibody for ST2/IL-1 R4 (AF523, R&D Systems, Minneapolis, USA), rabbit polyclonal antibody for ST2/IL-1 R4 (11920-1-AP, Proteintech Group, Chicago, USA), rabbit polyclonal antibody for TIMP1 (WL00869, Wanleibio, China), rabbit polyclonal antibody for TIMP2 (WL01209, Wanleibio, China), β-actin (TA-09, ZSGB-BIO, China), goat anti-mouse secondary antibody (ZB-2305, ZSGB-BIO, China), goat anti-rabbit secondary antibody (ZB-2301, ZSGB-BIO, China), and rabbit anti-goat secondary antibody (ab6741, Abcam, Cambridge, MA, USA).
8
Immunohistochemical Analysis of Cell Signaling
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The tissue sections were deparaffinized in xylene and dehydrated in ethanol (Sigma-Aldrich). Following dehydration, endogenous peroxides were inhibited, the sections were incubated with the goat polyclonal GSK-3β (sc8257), β-catenin (sc-1496) and rabbit polyclonal cyclin D1 (sc-753) primary antibodies and poly-HRP anti-goat (ab6741) or anti-rabbit IgG (ab6721; Abcam) secondary antibodies, and then incubated with 3,3′-diaminobenzidine (Sigma-Aldrich). The sections were then counterstained and dehydrated. Antibodies against GSK-3β, β-catenin and cyclin D1 were purchased from Santa Cruz Biotechnology, Inc.
9
Quantifying Antigen-Specific Antibody Responses
10
Evaluating Cell Proliferation with MTT and BrdU
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Cell proliferation was assessed using an MTT assay. Following transfection, A2780 cells were tested using the MTT assay at different time points. A total of 20 µl of 5 mg/ml MTT solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well and 150 µl dimethyl sulfoxide was added then incubated for 4 h at 37°C. Following this, the absorbance was measured using a microplate reader at a wavelength of 490 nm.
Cell growth was measured using a BrdU incorporation assay (EMD Millipore, Billerica, MA, USA). Following transfection and culturing for 24 h, A2780 were incubated with BrdU for 1 h, and stained with BrdU antibodies (cat. no. 61273; 1:500; Upstate Biotechnology, Inc., Lake Placid, NY, USA) at 4°C overnight, and then incubated with horseradish peroxidase-modified secondary antibodies (ab6741; 1:5,000; Abcam, Cambridge, UK) at 37°C for 2 h. Gray level images were measured under a laser-scanning microscope (Axioskop 2 plus; Carl Zeiss Co., Ltd., Jena, Germany).
Cell growth was measured using a BrdU incorporation assay (EMD Millipore, Billerica, MA, USA). Following transfection and culturing for 24 h, A2780 were incubated with BrdU for 1 h, and stained with BrdU antibodies (cat. no. 61273; 1:500; Upstate Biotechnology, Inc., Lake Placid, NY, USA) at 4°C overnight, and then incubated with horseradish peroxidase-modified secondary antibodies (ab6741; 1:5,000; Abcam, Cambridge, UK) at 37°C for 2 h. Gray level images were measured under a laser-scanning microscope (Axioskop 2 plus; Carl Zeiss Co., Ltd., Jena, Germany).
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