Scaffolds with differentiated cells or undifferentiated monolayer cells were washed with cold PBS, ground and total proteins were extracted by RIPA lysis buffer containing protease inhibitors. Protein concentration was evaluated by the Bradford procedure (Bio-Rad SA, Rosedale, New Zealand). Proteins lysates (20 μg) were separated on 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Non-specific binding sites of membranes were blocked with 10% non-fat milk powder in Tris-buffered saline containing 0.1% Tween (TBST, Sigma-Aldrich) for 1 h. Then, the blots were incubated overnight at 4 °C with anti-type I collagen (Novotec, 1:3000, Lyon, France), or anti-type II collagen (Novotec, 1:1500), or anti-type X collagen (Sigma, 1:5000), or rabbit anti-HtrA1 (Millipore, 1:2500). The membranes were also reacted with rabbit anti-GAPDH (Santa Cruz Biotechnology, Inc., 1:5000, Dallas, TX, USA) to verify equal loading. The next day, membranes were washed with TBST and, then incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch, 1:5000, West Grove, PA, USA) for 1 h at room temperature with 3% milk in TBST. Bound antibodies were detected using a chemiluminescence assay (Western Lightning® Plus-ECL, PerkinElmer, Inc., Waltham, MA, USA) and exposed to X-ray film for visualization.
Hrp conjugated goat anti rabbit or anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
HRP-conjugated goat anti-rabbit or anti-mouse IgG is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and visualize primary antibodies raised in rabbit or mouse.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
West femto maximum sensitivity substrate kit, by Thermo Fisher Scientific (2 mentions)
Bradford procedure, by Bio-Rad (1 mentions)
Tbs t, by Merck Group (1 mentions)
Rabbit anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
Cyclin a, by Santa Cruz Biotechnology (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
P stat3 s727, by Cell Signaling Technology (1 mentions)
Multigauge program, by Fujifilm (1 mentions)
P stat3 y705, by Cell Signaling Technology (1 mentions)
Las 4000 mini, by GE Healthcare (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
11 protocols using hrp conjugated goat anti rabbit or anti mouse igg
1
Quantification of Collagen and HtrA1 Proteins
2
Western Blot Analysis of Signaling Pathways
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Cell lysates were prepared in RIPA lysis buffer containing a protease inhibitor cocktail (Roche Diagnostics). Proteins (20‐50 μg) were resolved by 8%‐15% SDS‐PAGE and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dry milk or 5% BSA in TBST and incubated with primary and secondary antibodies according to the manufacturer's protocol. The antibodies used were p‐STAT3 (Y705) (Cell Signaling), p‐STAT3 (S727) (Cell Signaling), STAT3 (Cell Signaling), p‐ERK1/2 (T202/Y204) (Cell Signaling), anti‐ERK1/2 (Cell Signaling), Cyclin D1 (Santa Cruz), Cyclin A (Santa Cruz), BCl‐2 (Cell Signaling), Mcl‐1 (Santa Cruz), Survivin (Cell Signaling), Bcl‐xL (Santa Cruz), PARP (Cell Signaling) and GAPDH (Santa Cruz). The secondary antibodies used were HRP‐conjugated goat anti‐rabbit or anti‐mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The membranes were washed 3 times with TBST and then detected with the Luminata Forte Western HRP substrate (EMD Millipore) using the LAS 4000 mini (GE Healthcare Life Sciences). The densitometric analysis of the bands was performed using the MultiGauge program (Fuji Photo Film, Tokyo, Japan) and the results were normalized to the corresponding GAPDH.
3
Protein Expression Analysis in Cells
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Individual groups of cells were lyzed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor (Roche, CA, USA). Tissue samples were homogenized and lyzed in T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific), followed by sonication using the Vibra-Cell™ Ultrasonic Liquid Processors (Sonics & Materials). After being centrifuged, the concentrations of total proteins in individual samples were measured by a bicinchoninic acid assay (BCA). The lysate samples (50µg/lane) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk in TBST and probed with anti-HLA class I(1:1000, Abcam), anti-PD-L1 (1:1000, Cell Signaling Technology) and anti-GAPDH (1:5000, Abcam) at 4℃ overnight. After being washed, the membranes were incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories) and visualized with the enhanced chemiluminescent reagents (Thermo Fisher Scientific). The bands were analyzed by Alpha Imager (Alpha Innotech, San Leandro, CA, USA).
4
Quantification of protein targets by Western blot
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48 h after transfection with siRNA, cells were lysed in PBS containing 1% Triton X-100, 0.1% SDS, and protease inhibitors (6 µg/ml chymostatin, 0.5 µg/ml leupeptin, 10 µg/ml antipain, 2 µg/ml aprotinin, 0.7 µg/ml pepstatin, and 10 µg/ml amidinophenyl-methanesulfonyl fluoride; Sigma-Aldrich). The protein concentration of lysates was determined by using the BCA protein assay (Pierce; Thermo Fisher Scientific) with BSA as a standard. Cell lysates were then separated by 8% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% milk in PBS containing 0.1% Tween and probed with rabbit sera against APPL1, EEA1 (Miaczynska et al., 2004 (link)), and Rab5 (Abcam) or mouse monoclonal antibody against clathrin heavy chain (BD Biosciences). After washing, membranes were incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories), washed in PBS containing 0.1% Tween, incubated with electrochemiluminescence reagent, and exposed to an x-ray film.
5
Immunoblot Analysis of Protein Expressions
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Protein was extracted from cells by using Radio-Immunoprecipitation Assay buffer (Suolaibo Biotechnology Co. Ltd, Shanghai, China) containing complete protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany), and was then separated through 10% sodium dodecyl sulfate polyacrylamide gel, transferred onto PVDF membrane (Millipore, Billerica, CA), probed with a rabbit polyclonal antibody against ISL1 protein (1:8000 dilution; Abcam, Cambridge, UK), or a rabbit monoclonal antibody against EZH2 (1:1000 dilution; Epitomics, Burlingame, CA), or a mouse polyclonal antibody against GAPDH (1:105 dilution, Roche), followed by incubation with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibody. Signals were visualized by using the method of chemiluminescence (Millipore).
6
Actomyosin Concentration Analysis
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Protein concentration of actomyosin was determined at OD 595, using BSA as standard. 2.0 μg of purified actomyosin were electrophoresed on NuPAGE 4–12% gels (Invitrogen) and wet-blotted to PVDF membranes overnight at 30 mA. Membranes were probed with specific antibodies at the dilutions listed above, followed by HRP-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories). Signals were detected with a WestFemto maximum sensitivity substrate kit (Thermo Scientific). Immunoblots were photographed using a BioRad Chemidoc PM Molecular Imager and band densities were measured using ImageJ64 software. Montages were assembled using Adobe Illustrator, and then converted to TIFF files. Full gel scans can be found in Supplementary Figs 11–13 .
7
Protein Quantification and Western Blot
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Protein concentrations were determined as OD 595, using BSA as standard. 10 µg of soluble protein (100 V, 60 min) was electrophoresed on 12% SDS–polyacrylamide gels or gradient gels (4–20%, BIORAD), and wet-blotted for 3 h at 50 mA. Membranes were probed with specific antibodies at the dilutions listed above, followed by HRP conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories). Signals were detected with a West Femto maximum sensitivity substrate kit (Thermo Scientific) on blue films (Midsci).
8
Western Blot Protein Analysis
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Protein samples from cells were analyzed by SDS-PAGE and transferred to a nitrocellulose membrane (Bio‐Rad, Hercules, CA, USA). The membrane was blocked with 5% milk for 1 h at room temperature, followed by incubation with the indicated primary antibodies at 4 °C overnight. The membrane was subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-Mouse or anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Proteins were detected using Image Quant LAS4000 (GE Healthcare, Chicago, IL, USA).
9
Western Blot Analysis of P2X7R in Rat Retinas
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Western blot analysis was conducted as previously described with some modifications [3 (link)]. After washed with PBS solution, rat retinas or cells were lysed in lysis buffer (containing 1 M Tris-HCl at pH 7.5, 1% Triton X-100, 1% Nonidet p-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined by the BCA method (Pierce). Protein samples (1.0 g/L, 15 L) were subjected to 10% SDS-PAGE using a Mini-Protean 3 electrophoresis system (Bio-Rad) and electrotransferred to polyvinylidene fluoride membranes using a Mini TransBlot electrophoretic transfer system (Bio-Rad). The membranes were blocked with 5% skimmed milk at room temperature for 1 h and then incubated with rabbit antibody against-P2X7R (1 : 1000, Abcam). The blots were washed with TBST and incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG (1 : 4000, Jackson ImmunoResearch Laboratories) for 1 h at room temperature and visualized with enhanced chemifluorescent reagent ECL (Thermo Scientific, Rockford, IL, USA) and exposed to X-ray film in the dark. The experiments were performed in triplicate, and the protein bands were quantitatively analyzed with Image J Analysis software.
10
Western Blot Analysis of Cytoskeletal Proteins
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The cells were cultured in a 10-cm dish, and after the removal of the cell culture medium, 100 μl ice-cold PBS containing 0.5% protease inhibitor III (Calbiochem), 0.5% protease inhibitor VI (Calbiochem), and 1 mM EDTA was added, and then the cells were extracted with 25 μl of 5x sample buffer (312 mM Tris-HCl, pH 6.8, 10% SDS, 35% glycerol, 0.05% bromophenol blue, and 25% 2-mercaptoethanol). The whole extract was placed in boiling water for 1 min and centrifuged for 1 min, and the supernatant was subjected to polyacrylamide gel electrophoresis. The proteins were then electro-transferred to a polyvinyl difluoride membrane using a transfer apparatus, according to the manufacturer’s protocol (Bio-Rad). After an incubation with 3% skim milk in 150 mM NaCl buffered with 10 mM Tris-HCl (pH 7.4) for 30 min at room temperature, the membrane was incubated with 1 μg/ml antibody IgG against filamin A (rabbit, Cell Signaling Technology), Halo-tag protein (rabbit, Promega), α-tubulin (mouse, Abcam), or β-actin (mouse, Sigma-Aldrich) for 1 h, followed by an incubation with HRP-conjugated goat anti-mouse or anti-rabbit IgG (1:10,000; Jackson Immunoresearch Laboratories) for 1 h. The bands of the labeled proteins were then visualized using the Western BLoT substrate series (TAKARA BIO), according to the manufacturer’s protocol.
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