Cellular proteins were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Complete Protease Inhibitor; Roche, Manheim, Germany). Proteins were quantified by bicinchoninic acid assay (Thermo Fischer Scientific). 20 μg of proteins from each sample were separated by SDS polyacrylamide gel electrophoresis on a 12% gel in MOPS running buffer (ThermoFisher Scientific), transferred onto a nitrocellulose membrane, and probed with primary antibodies against PROX1, CD31, OCT3/4, and β-actin (Abcam). After incubation in HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Invitrogen), bound antibodies were detected using FluorChem E (Protein Simple). Protein expression was analyzed by densitometry using ImageJ, and normalized to the house-keeping β-actin.
Hrp conjugated goat anti mouse or anti rabbit secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies are used to detect and amplify the signal of primary antibodies in immunoassays and other applications. They bind to the constant region of mouse or rabbit primary antibodies and are conjugated with horseradish peroxidase (HRP) enzyme for colorimetric or chemiluminescent detection.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid assay, by Thermo Fisher Scientific (3 mentions)
Mops running buffer, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor, by Roche (3 mentions)
Rabbit anti collagen 4, by Abcam (2 mentions)
Mouse anti fibronectin, by Abcam (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Hybond p pvdf membrane, by GE Healthcare (2 mentions)
Hybond, by Cytiva (2 mentions)
Mouse anti gapdh, by Abcam (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Mouse anti αb crystallin, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Ab216347, by Abcam (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ne pertm nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Ab8227, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
6 protocols using hrp conjugated goat anti mouse or anti rabbit secondary antibodies
1
Protein Expression Analysis by Western Blot
2
Validation of LDB3 Antibody
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Primary antibodies are listed in Supplementary Table 3 . Antibodies against LDB3 were generated by immunizing rabbits with a peptide corresponding to amino acid residues 116–130 encoded by exon 6 of human LDB3 (NP_001073585; LDB3ex6ab; Supplementary Fig. 5a ). We validated the LDB3ex6ab antibody for immunoblotting and immunofluorescence using transfected COS-7 cells and tissues of Ldb3+/+ and Ldb3−/− mice (Supplementary Fig. 5b–d ). Alexa Fluor-, IRDye-, and HRP- conjugated goat anti-mouse or anti-rabbit secondary antibodies were purchased from Invitrogen, LI-COR biosciences, and Jackson ImmunoResearch Laboratories, respectively.
3
Protein Extraction and Western Blot Analysis
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After treatments, cellular proteins were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCL, pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 25 mM NaF, 0.1 mM sodium orthovanadate, 10 mM NaP4O7, 1 nM phenylmethyl sulfonyl fluoride) containing protease inhibitors (Complete Protease Inhibitor; Roche, Manheim, Germany) on ice. Total protein concentrations were quantified by a bicinchoninic acid assay (Thermo Fischer Scientific, Waltham, MA, USA). A total of 10 μg of proteins from each sample were separated by SDS polyacrylamide gel electrophoresis on a 4% to 12% gel in MOPS running buffer (Life Technologies, Carlsbad, CA, USA), transferred onto a polyvinylidene fluoride (PVDF) membrane and probed with the following primary antibodies: rabbit anti-MMP14, mouse anti-fibronectin, rabbit anti-collagen IV, and mouse anti-GAPDH (Abcam, Boston, MA, USA). HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used. Bound antibody complexes were detected using FluorChem E (ProteinSimple, San Jose, CA, USA). Protein expression was analyzed by densitometry using ImageJ (National Institutes of Health [NIH], Bethesda, MD, USA), and normalized to the housekeeping protein, GAPDH. All experiments were performed in triplicate for each of four donor cells.
4
Quantitative Protein Expression Analysis
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Cellular proteins were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCL, pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 25 mM NaF, 0.1 mM sodium orthovanadate, 10 mM NaP4O7, 1 nM phenylmethyl sulfonyl fluoride) containing protease inhibitors (Complete Protease Inhibitor, Roche, Manheim, Germany) on ice. Proteins were quantified by bicinchoninic acid assay (Thermo Fischer Scientific). 20 μg of proteins from each sample were separated by SDS polyacrylamide gel electrophoresis on a 4–12% gel in MOPS running buffer (ThermoFisher Scientific), transferred onto a PVDF membrane and probed with the following primary antibodies rabbit anti-myocilin (Sigma Aldrich), mouse anti-αB-crystallin, mouse anti-fibronectin, rabbit anti-collagen IV and mouse anti-β-actin (Abcam). HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Invitrogen) were used. Bound antibody was detected using FluorChem E (Protein Simple). Protein expression was analyzed by densitometry using ImageJ, and normalized to the housekeeping gene β-actin. All experiments were performed in duplicate for each of three donor cells.
5
Extraction and Analysis of Cellular Proteins
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Total cytoplasmic and nuclear proteins from OVCAR-3 cells were extracted with RIPA buffer (Thermo Fisher, USA). NE-PERTM Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher, USA) was added with protease and phosphatase inhibitor cocktail (Thermo Fisher, USA). BCA protein assay kit (Beyotime Biotechnology, China) was used for quantification. Protein (30 μg) was loaded onto SDS-PAGE gel and transferred to a Hybond-P PVDF membrane (GE Healthcare, USA). 5% fat-free milk in TBST (Tris-buffered saline with 0.1% Tween 20) was used for blocking. The following antibodies were incubated with the membranes at 4°C overnight: mouse anti-E-cadherin (1 : 2000, CST: 3195, USA), rabbit anti-vimentin (1 : 2000, CST: 5741, USA), rabbit anti-Snail (1 : 1000, Abcam: ab216347, USA), and actin (1 : 5000, Abcam: ab8227, USA) as an internal control. Protein expression was visualized on X-ray films using the HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1 : 5000, Thermo Fisher, USA) and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher, USA). Band intensities were quantitated using Image Pro Plus 6.0 software. The results were presented as the density ratio of the target protein band to the internal control.
6
Western Blot Analysis of DAOY, UW228, and UW426 Cells
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