Proteins were separated on 10% SDS-PAGE and transferred to Immobilon-P PVDF membrane (EMD Millipore). Membranes were probed with primary antibodies, followed by light chain specific HRP-conjugated secondary antibodies at 1:5000 (Bio-Rad) and developed using Immobilon Western HRP substrate (EMD Millipore). EGF levels were detected by dot plots. Conditioned media from transfected 293T cells were concentrated 15x using 3kDa ultrafiltration tubes (Amicon, EMD Millipore). Serial dilution of recombinant EGF protein was used as a standard curve. 2uL of non-denatured protein preparation was loaded directly onto 0.2μm nitrocellulose membrane (GE Life Sciences) and allowed to dry. Membranes were blocked in 5% BSA in TBST for 1h and probed with rabbit anti-EGF (cat# ab9695, Abcam), followed by HRP-conjugated secondary antibody at 1:5000 (Bio-Rad) and developed using Immobilon Western HRP substrate (EMD Millipore). Membranes were visualized using FluorChem HD2 (Alpha Innotech) or exposed to BIOMAX film (Eastman Kodak).
Hrp conjugated secondary antibody
Manufactured by Bio-Rad
Sourced in United States, France, Italy, Spain, Japan, Germany, Canada, United Kingdom
HRP-conjugated secondary antibodies are a type of enzyme-labeled antibodies used in various immunoassays and detection techniques. They are designed to bind to primary antibodies and catalyze a colorimetric or chemiluminescent reaction, enabling the visualization and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (18 mentions)
Pvdf membrane, by Merck Group (14 mentions)
Pvdf membrane, by Bio-Rad (12 mentions)
β actin, by Merck Group (11 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Clarity western ecl substrate, by Bio-Rad (10 mentions)
Chemidoc imaging system, by Bio-Rad (9 mentions)
β actin, by Cell Signaling Technology (9 mentions)
Nitrocellulose membrane, by GE Healthcare (8 mentions)
Protease inhibitor cocktail, by Merck Group (7 mentions)
Ripa buffer, by Merck Group (7 mentions)
Quantity one software, by Bio-Rad (7 mentions)
Chemidoc mp imaging system, by Bio-Rad (7 mentions)
Ecl substrate, by Bio-Rad (6 mentions)
Gapdh, by Santa Cruz Biotechnology (6 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (5 mentions)
Bradford assay, by Bio-Rad (5 mentions)
Protease inhibitor, by Roche (5 mentions)
Bca assay, by Thermo Fisher Scientific (4 mentions)
Image lab software, by Bio-Rad (4 mentions)
267 protocols using hrp conjugated secondary antibody
1
Western Blot and Dot Blot Analysis of EGF
2
Protein Extraction and Analysis from Ocular Tissues
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Whole eye and dissected iris, cornea, retina, optic nerve, lens, lens epithelium, cortex and nucleus were immediately homogenised in Laemmli 1× buffer and boiled at 95°C for 5 min for protein analysis. The samples were sonicated and the supernatants stored at −80°C. Cortex and nucleus were separated from lens mass and homogenised in Tris/HCl 10 mM pH 7.4 + PMSF + PIC (Sigma–Aldrich) and sonicated. Then the samples were centrifuged at 100000×g for 10 min at 4°C. The resulting pellets were resuspended in the same buffer and centrifuged at 10000×g for 10 min at 4°C. The pellets were then dissolved in Urea 7 M (Sigma–Aldrich) in 10 mM Tris/HCl pH 7.4 and centrifuged twice at 100000×g for 10 min at 4°C. The final pellets were dissolved in Tris/HCl pH 7.4 10 mM, sonicated and stored at −80°C [20 (link)].
Extracted proteins were subjected to Western blot analysis with anti-HOPS polyclonal antibody [2 (link)] and the appropriate HRP-conjugated secondary antibody (Bio-Rad, Hercules, CA, U.S.A.) and visualised with ECL (GE Healthcare Life Sciences, Little Chalfont, U.K.).
Extracted proteins were subjected to Western blot analysis with anti-HOPS polyclonal antibody [2 (link)] and the appropriate HRP-conjugated secondary antibody (Bio-Rad, Hercules, CA, U.S.A.) and visualised with ECL (GE Healthcare Life Sciences, Little Chalfont, U.K.).
3
SDS-PAGE and Western Blot for Protein Analysis
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Cells were grown on a six-well plate, trypsinized, washed with PBS, and resuspended 50 µl PBS. This solution was mixed with 34 µl of 10% SDS and 1 µl of 1 M DTT, boiled for 5 min, and cooled to 23°C. 17 µl of 300 mM freshly made NEM in water was added. Just before SDS-PAGE, the protein sample was mixed 1:1 with buffer containing 250 mM Tris-HCl, pH 6.8, 2 mM EDTA, 20% glycerol, 0.8% SDS, 0.02% bromophenol blue, 1,000 mM NaCl, and 4 M urea. Proteins were separated by 7.5% SDS-PAGE and transferred to a PVDF membrane (Millipore). The membrane was blocked with TBS-T (20 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.1% Tween-20) containing 3% BSA (Research Organics) for 1 h, then incubated with the primary antibody solution at 4°C overnight. After washing with TBS-T, the membrane was incubated with HRP-conjugated secondary antibody (Bio-Rad) for 1 h at room temperature. Signals were detected by chemiluminescence (Pierce). For Western blot of Mff KO cells, the Odyssey CLx system was used (Li-Cor Biotechnology), as well as IRDye-labeled anti-rabbit and anti-mouse secondary antibodies from the same company.
4
Western Blot Protocol for Protein Detection
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Cell lysates or IPs were resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA). Membrane was blocked with 5% milk-TBST and probed with the primary antibody (1:1,000 dilution), HRP-conjugated secondary antibody (1:3,000 dilution), and ECL reagents (Bio-Rad).
5
Western Blot Analysis of Cell Signaling
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Total protein lysates were obtained using RIPA buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 50 mM Tris (pH 7.6), and 10 μL/mL protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Equal amounts of protein from each sample were separated on SDS-PAGE gels and then transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk or 5% bovine serum albumin in TBS-T (150 mM NaCl, 10 mM Tris, pH 7.4, and 0.1% Tween-20), and incubated with the following primary antibodies at 4°C overnight: poly [ADP-ribose] polymerase (PARP), cleaved PARP, CHK1, phosphorylated CHK1 (Ser345), pH2A.X, and Rad51 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-CDK 4, anti-cyclin A, -B1, -D1, -E, p21CIP1, and cdc25A (1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), active caspase 3 (1:500; Epitomics, Burlingame, CA), and anti-actin (1:3000; Millipore, Billerica, MA, USA) as a loading control. After several washes with TBS-T, the membranes were incubated with HRP-conjugated secondary antibody (1:5000; Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence detection system (GE Healthcare, Wauwatosa, WI, USA).
6
Comprehensive Protein Expression Analysis
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We used a 10% SDS-PAGE gel to separate the proteins and transfer them to a PVDF membrane. We used the following antibodies to probe NEK2, p21, p27, CDK2, CDK4, CDK6, cyclinD1, E-cadherin, N-cadherin, Vimentin, MMP9, MMP2, p-GSK3β (Ser9), t-GSK3β, β-catenin, c-Myc, IL-10, and GAPDH at 4 °C for 12 h and incubated with HRP-conjugated secondary antibody for 2 h following detection by chemiluminescence (Bio-Rad Laboratories, Inc., USA). Antibodies used for immunoblotting are listed in Table S3 .
7
Quantifying Amyloid Precursor Protein Fragments
8
Western Blot Analysis of Mitochondrial Proteins
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Equal amounts of denatured mitochondrial protein were loaded on each lane, and the proteins were resolved on precast polyacrylamide gels (BioRad, Hercules, CA), under reducing or non-reducing conditions, and subsequently transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were blocked in 5% milk prepared with 0.1% Tween 20 in TBS at room temperature for 1 h. Anti-3-nitrotyrosine (1:3000) (Cell Signaling, Danvers, MA), anti-VDAC (1:3000) (Cell Signaling), anti-acetyl-lysine (1:3000) (Cell Signaling), and CypD (1:5000) (Cell Signaling) were used as primary antibody. The protein bands were visualized by enhanced chemiluminescence (Millipore) after incubation with HRP-conjugated secondary antibody (1:10,000) (BioRad). anti-VDAC antibody was used as loading control.
9
Capsule Detection via Blotting Assay
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Capsule presence was detected using the capsule blotting method [27 (link)]. Briefly, strains were grown to mid-logarithmic phase (OD620 = 0.4) in C+Y and were lysed using DOC/SDS disruption of the membrane. Proteins were removed by incubation with proteinase K; a small sample of the cell lysate was used to measure protein concentration prior to protein degradation via BCA assay (Pierce). Cell lysates were diluted to 22.5 mg/mL cellular protein and subjected to agarose gel electrophoresis. The samples were transferred to a nitrocellulose membrane following standard capillary transfer in a high-salt buffer. The membrane was then probed with serotype-specific antibodies (Statens Serum Institute) and HRP conjugated secondary antibody (Bio-Rad), followed by detection using a HRP chemiluminescent substrate (Thermo Scientific).
10
Protein Extraction and Western Blot Analysis
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Cell pellets were suspended in RIPA buffer (5 mM EDTA, 0.15 mol NaCl, 0.1 mol Tris pH 8.0, 1% Triton) with protease and phosphatase inhibitors cocktail (10 μg/mL; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and sonicated three times (for 10 s and 70% amplitude). The samples were incubated on ice for 30 min, and then centrifuged at 10,000× g for 20 min to remove pellet residues. Equal amounts of protein (30 μg/lane) estimated by Bradford protein assay were solubilized in a 2X Laemmli sample buffer containing 20% of 2-mercaptoethanol. Proteins were separated by SDS-PAGE on 10–12% polyacrylamide gel and then electro-transferred to nitrocellulose membranes (Trans-Blot Turbo Mini Nitrocellulose, Bio-Rad, Hercules, CA, USA). Membranes were, then, incubated overnight at 4 °C with rabbit polyclonal anti-α-actin (Abcam, Cambridge, UK) and with mouse monoclonal anti-vinculin (Santa-Cruz Biotechnologies, Dallas, TX, USA). Thus, membranes were incubated for 1 h with HRP-conjugated secondary antibody (1:3000; Bio-Rad, CA, USA). The immune complexes were detected by enhanced chemiluminescence substrate (ECL Substrates, Bio-Rad, CA, USA). Densitometric analysis of the bands was performed using Image J software. The results were expressed as arbitrary unit (A.U.) and represented as mean of three independent experiments.
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