Protein lysates were prepared in a lysis buffer [5 mmol/L MgCl2, 137 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% CHAPS, 10 mmol/L HEPES (pH 7.5)] supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail 2 (both from Sigma, St. Louis, MO), and then normalized using NanoDrop measurement (Thermofisher scientific, Waltham, MA) or Bio-Rad protein assay (Hercules, CA, catalog no. 500–0006). After being denatured in LDS sample buffer (Invitrogen) supplemented with 2-Mercaptoethanol (Bio-Rad, Hercules, CA), protein samples were loaded onto 10% or 14% SDS-PAGE gels which were then transferred electrophoretically onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 0.2% I-Block (Applied Biosystems, Grand Island, NY) in PBS-T (PBS containing 0.1% Tween 20) and incubated with the primary antibodies in PBS-T containing 0.2% I-Block overnight at 4°C or at room temperature for 3 hours. The membranes were then washed and incubated with a secondary antibody in PBS-T containing 0.2% I-Block conjugated to alkaline phosphatase, followed by development with CDP-Star substrate (Applied Biosystems, Grand Island, NY).
I block
Manufactured by Thermo Fisher Scientific
Sourced in United States
The I-Block is a compact and versatile laboratory equipment designed for incubation and temperature control applications. It provides a consistent and accurate temperature environment for various sample types, enabling efficient and reliable experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Iblot transfer system, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Pvdf membrane, by Roche (3 mentions)
Phosstop, by Roche (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Bio-Rad (2 mentions)
Nanodrop measurement, by Thermo Fisher Scientific (2 mentions)
Lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Cdp star substrate, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (2 mentions)
I block, by Jackson ImmunoResearch (2 mentions)
73 protocols using i block
1
Western Blot Analysis of Protein Lysates
2
Western Blot Analysis of Protein Lysates
3
Serological Detection of Relapsing Fever Spirochetes
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Western blot analysis was performed to detect antibodies to relapsing fever spirochetes using a whole-cell lysate of B. hermsii DAH and a purified recombinant GlpQ protein from the same strain [18 (link)]. The protein preparations were separated by electrophoresis in Novex® 4–20 % Glycine Gels 1.0 mm (Invitrogen, Life Technologies, Grand Island, NY, USA), and transferred to nitrocellulose membranes using the iBlot® Gel Transfer Device according to the manufacturer’s instructions (Invitrogen, Life Technologies). Membranes were blocked in Tropix® I-BLOCK (Applied Biosystems, Life Technologies, Grand Island, NY, USA) at room temperature for 1 h. Serum samples were diluted 1:100 in 5 ml of I-BLOCK and incubated with the membrane at room temperature for 1 h. Membranes were removed from the serum samples, washed with I-BLOCK, and incubated with HRP-conjugated recombinant Protein A (1:4,000) (Invitrogen, Life Technologies). The membranes were washed in I-BLOCK for 2 h with four changes of the wash solution, and then developed for ~30 s using ECL® Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK). Positive and negative control samples were obtained from infected and uninfected laboratory mice. A serum sample was considered positive if it contained antibodies that bound to eight or more proteins in the B. hermsii whole-cell lysate and to the purified GlpQ.
4
Western Blot Immunodetection Protocol
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Proteins were separated by SDS-PAGE gel electrophoresis and transferred to a 0.45-μm nitrocellulose membrane (Bio-Rad) according to standard procedures. The membrane was blocked with 5% milk or with 0.2% I-block (Invitrogen) in PBST (PBS, 0.1% Tween-20) for 1 hour. The membrane was incubated with primary antibodies for 2 hours at room temperature or overnight at 4°C, followed by 3x washes for 5 min in PBST and incubation with secondary antibodies for 1 hour at room temperature. The membrane was washed three times for 5 min with PBST and then imaged with Odyssey system (Li-Cor). When primary antibody was HRP-conjugated, the membrane was washed 3× 5 min with PBST, incubated with the HRP substrate, and X-ray film developed on an X-Ray Film Processor (Konica Minolta). The following antibodies were used: HRP-conjugated anti-FLAG (Sigma, A8592), mouse anti-FLAG (Sigma, F1804), rabbit polyclonal anti-GFP (Chen et al., 2016 (link)), IRDye anti-rabbit and anti-mouse secondary antibodies (Li-Cor, #925–68070 and #925–32211).
5
Western Blot Analysis of Protein Samples
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After washing with DPBS (GIBCO), cells were either directly boiled (95°C) in sample buffer (200 mM Tris–HCL, 6% SDS, 20% glycerol, 300 mM DTT and bromophenol blue) or were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 1× protease inhibitors [Roche], and 1× PhosStop [Roche]). Protein samples were loaded on an SDS–PAGE gel and transferred on a nitrocellulose or PVDF membrane (2 h 15 min, 0.3A). Immunoblots were blocked in 5% milk in TBS-T (TBS with 0.1% Tween20) or in I-Block (Invitrogen). Membranes were incubated overnight at 4°C with the primary antibody. After three washing steps with TBS-T, secondary HRP-coupled antibodies were added for 1 h, and then membranes were washed again with TBS-T. Finally, membranes were covered with ECL solution (PerkinElmer), and chemiluminescence was measured. Immunoblot quantification was performed in ImageJ using the “Gel Analysis” tool for measurement of the blot band density.
6
Western Blot Analysis of Protein Samples
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Cells were washed with DPBS before harvesting by scraping on ice. Lysis was performed with RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium desoxycholate, 1% Triton X, PhosStop [Roche], and protease inhibitor [Roche]) and protein concentrations were adjusted using BCA assays. Protein samples were separated by SDS–PAGE (100V) and transferred (2 h 15 min, 0.3 A) on a nitrocellulose membrane (0.45 μm pore size). Membranes were blocked with I-Block (Invitrogen) and incubated at 4°C overnight with the primary antibody. After washing three times with TBS-T-buffer, secondary antibody coupled to horseradish peroxidase was added to the membrane for 1 h at RT. Immunoblots were washed another three times with TBS-T before enhanced chemiluminescence analysis using ECL (PerkinElmer) and x-ray films (Fuji Medical).
7
Western Blot for Phospho-Syk and Total Syk
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Cells were treated with compounds for 6 h, and whole-cell lysates were separated on 4–12% SDS–PAGE precast gels (LifeTech). Proteins were transferred to PVDF membranes and subsequently blocked with iBlock (Invitrogen) for 30 min. Then primary antibody was added overnight at 4°C, and after washing with PBS with 1% Tween (PBS-T), secondary HRP-coupled antibody was incubated for 45 min. Blots were once again washed with PBS-T and soaked with ECL substrate (Western Lightning Plus-ECL), and signals were detected with an imaging system (MF-ChemBIS 3.2; DNR Bio-Imaging Systems). Specific proteins were detected using anti-phospho SYK (Cell Signaling Technology #2711) or total SYK protein (Cell Signaling Technology #2712).
8
GFP Fusion Protein Validation
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Similar cultures as analyzed by fluorescence microscopy were harvested to test for the stability and correct expression of the GFP fusion proteins. Cell cultures were harvested at 5000 rpm for 20 min, and the pellet was resuspended in 1x SDS buffer (250 mM Tris pH 6.8, 10% SDS(w/v), 10% Dithiothreitol (DTT) (w/v), 50% glycerol (v/v), 50 µg/mL) concentrated in PBS (Phosphate Buffered Saline) to a theoretical O.D of 22. 10µL of the sample was loaded and run on a 11% SDS-PAGE. After gel electrophoresis, the proteins were transferred to a PVDF membrane using semidry blotting in blotting buffer (5 mM Tris, 40 mM glycine, 20 (v/v), 0.0375% (v/v)). The membrane was blocked with 0.1 % I-Block TM (Applied Biosystems, California USA) for 1 hour at room temperature. Next, the membrane was incubated in primary antibody, α GFP (Sigma-aldrich, California USA; 1:5000 dilution) for 3-4 hours at 4ºC. After incubation, the membrane was washed 3 times with PBST. The secondary antibody, α-rabbit coupled to Horse Radish Peroxidase (from goat) (ThermoFischer Scientific, Masasachusetts USA; 1:10,000 dilution) was added and incubated for 1 hour at 4ºC. The membrane was washed 3 times with PBST after the incubation period, and prepared for development. Invitrogen TM I-Bright TM (ThermosFischer Scientific) scanner was used for image development.
9
Analyzing GFP Fusion Protein Stability
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Similar cultures as analyzed by fluorescence microscopy were harvested to test for the stability and correct expression of the GFP fusion proteins. Cell cultures were harvested at 5000 rpm for 20 min, and the pellet was re-suspended in 1x SDS buffer (250 mM Tris pH 6.8, 10% SDS(w/v), 10% Dithiothreitol (DTT) (w/v), 50% glycerol (v/v), 50 mg/mL) concentrated in PBS (Phosphate Buffered Saline) to a theoretical O.D of 22. 10mL of the sample was loaded and run on a 11% SDS-PAGE. After gel electrophoresis, the proteins were transferred to a PVDF membrane using semidry blotting in blotting buffer (5 mM Tris, 40 mM glycine, 20 (v/v), 0.0375% (v/v)). The membrane was blocked with 0.1% I-Block TM (Applied Biosystems, California USA) for 1 hour at room temperature. Next, the membrane was incubated in primary antibody, a GFP (Sigma-aldrich, California USA; 1:5000 dilution) for 3-4 hours at 4 C. After incubation, the membrane was washed 3 times with PBST. The secondary antibody, a-rabbit coupled to Horse Radish Peroxidase (from goat) (ThermoFischer Scientific, Masasachusetts USA; 1:10,000 dilution) was added and incubated for 1 hour at 4 C. The membrane was washed 3 times with PBST after the incubation period, and prepared for development. Invitrogen TM I-Bright TM (ThermosFischer Scientific) scanner was used for image development.
10
Western Blot Analysis of Cochlear Proteins
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Proteins from cochlear extracts were separated on 4–12% gradient NuPage gels (Invitrogen, Carlsbad, CA), transferred to polyvinylidene difluoride membranes, blocked with 0.1% I-Block (Applied Biosystems, Foster City, CA) and probed with Stat3 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) using chemiluminescence detection (Pierce Chemical Co., Rockford, IL). A Fuji model LAS 1000 imaging system (Stamford, CT) was used to visualize bands. Background corrected bands were normalized against bands obtained with actin antibodies [1] (link).
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