Western blotting was performed essentially as previously described (Thompson et al., 2022 ; Galea et al., 2020 ). Cells were lysed on ice for 30 min in 50 mm Tris Base pH 7.6, 150 mm NaCL, 1% Triton X-100, 0.02% Sodium Azide, 1 mm protease inhibitor cocktail (Merck Life Science UK Ltd), 1 mm sodium orthovanadate, 25 mm sodium fluoride. Protein concentration was determined using Pierce BCA Protein Assay kits (Thermo Fisher). Proteins were resolved by SDS-PAGE using 10% polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked in TBS (15.4 mm Trizma-HCL, 4.62 mm Tris-base, 150 mm NaCl, pH 7.6) containing 10% w/v milk powder (Merck Life Science UK Ltd). Membranes were incubated with primary antibody overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibodies (Agilent Technologies, Stockport, UK) for 1 h. Primary antibodies were as described in the immunofluorescence section. HRP was detected using ECL Prime (Cytiva, Amersham, UK). To strip and re-probe, membranes were incubated in 0.2 m NaOH for 20 min at 37 °C and then 20 min at room temperature before re-blocking and re-use.
Milk powder
Manufactured by Merck Group
Sourced in Germany, United States
Milk powder is a laboratory product that is produced by evaporating and dehydrating raw milk. It is a fine, powdery substance that can be easily stored and transported. The core function of milk powder is to provide a concentrated form of milk solids, which can be used in various laboratory applications and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (3 mentions)
Trans blot electrophoretic transfer cell, by Bio-Rad (2 mentions)
Hyperfilm ecl, by GE Healthcare (2 mentions)
Maf 55013 1 ap, by Proteintech (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Histone 3, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Fastblot b34 blotting device, by Analytik Jena (2 mentions)
Pefabloc sc, by Merck Group (2 mentions)
Fusion fx7 detection system, by Vilber (2 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx any kd precast gels, by Bio-Rad (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Tbs t, by Merck Group (1 mentions)
Tbs t, by Agilent Technologies (1 mentions)
Tween 20 tbst, by Merck Group (1 mentions)
18 protocols using milk powder
1
Western Blotting Protocol for Protein Analysis
2
Western Blot Analysis of LC3 Protein
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Cells were harvested in LIPA buffer (Sigma) as previously reported13 (link). After centrifugation at 15,000 × g for 5 min, the supernatant was boiled for 5 min in SDS-PAGE sample buffer (Wako) and loaded onto 5–20% gradient or 15% gels for 1~2 h. Proteins were transferred to nitrocellulose membranes (Bio-Rad) by using the Trans-Blot Electrophoretic Transfer cell (Bio-Rad). The membrane was washed in 20 mM Tris-HCl buffer with 1% Tween 20 (TBS-T) (Sigma-Aldrich), incubated for 1 h in blocking buffer (5% milk powder (Merck) in TBS-T), and then further incubated overnight with primary antibodies; Anti-LC3 (Cell signaling) and anti-GAPDH (Santa Cruz) antibodies were purchased and diluted in blocking buffer by 1:3000. The horseradish peroxidase (HRP)-labeled secondary antibody (Dako) was diluted by 1:5000 in TBS-T for secondary antibody staining for 1 h at RT. The membrane was then washed in TBS-T and the signals were developed using the Lumi-Light reagent (Roche) for 5 min. The signal was detected on the LAS-3000 imaging system (Fuji).
3
Bacterial Growth and Enumeration
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A strain of B. cereus (26 B, own collection; isolated from raw cow milk) and a reference strain of S. aureus (Czech Collection of Microorganisms, Masaryk University, Brno; CCM 6188) were multiplied in a broth (10 g Pepton, 10 g Lab Lemco Poder, 5 g NaCl, 1000 ml distilled water) at 36 °C for 24 h. Two strains of P. fluorescens, ZB 66 (own collection; isolated from raw cow milk) and CCM 2826 (reference strain) were multiplied in a Pseudomonas broth containing 1% of glycerol and 0.1% of milk powder (Merck) at 30 °C for 24 h. Then the counts of bacteria (CFU/ml) were determined (ČSN EN ISO 7218, 2008) .
4
Immunoblotting Analysis of H7 Flagellin
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H7 flagellin was separated through a 12% SDS-PAGE as mentioned above and transferred onto Hybond ECL nitrocellulose membrane (GE Healthcare Life Sciences, Marlborough, MA, USA) using a Trans-Blot electrophoretic transfer cell (Bio-Rad). After membrane blocking with 5% (w/v) milk powder (Sigma) in PBS at 4°C overnight, it was sequentially incubated first with 1/2000 rabbit polyclonal anti-H7 flagellin (Abcam, Boston, MA, USA), 1/50000 plasma from H7-immunized BALB/c mice or 1/10000 plasma from control BALB/c mice, and second with 1/3000 HRP-conjugated goat polyclonal anti-rabbit IgG or anti-mouse IgG (Invitrogen, San Diego, CA, USA), all diluted in PBS containing 5% (w/v) milk powder. Incubations were carried out at room temperature for at least 1 h on a platform shaker and were washed 3 times during 5 min with 0.1% (v/v) Tween20 in PBS before and after each antibody step. Specific bands were visualized by enhanced chemiluminescence (ECL) method (GE Healthcare Life Sciences).
5
Western Blot Analysis of M. tuberculosis
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Concentrated supernatants from M. tuberculosis CDC1551, Δppe38–71, the ppe38–71 complemented strain, S507, S3651, S1453, S3760, S4437, S3839, S5218, H37Rv, S4570, S1116, S2701 and S2135 were separated by SDS-PAGE (12% resolving gel, 100 V, 1 h) and transferred to a nitrocellulose membrane. The membrane was blocked with 1% milk powder (Sigma-Aldrich, MO, USA) for 1 h and probed with either mouse monoclonal α-PGRS (1:5000) (41 (link)) and α-EsxA (1:500) (42 (link)) or rabbit polyclonal α-PPE38 (1:1000) (this study) overnight at 4°C. Primary antibodies were removed by washing with Tris-buffered saline supplemented with Tween (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) followed by probing with either goat anti-mouse or goat anti-rabbit horse radish peroxidase conjugated secondary antibody for 1 h. Probed nitrocellulose membranes were visualized using a ChemiDoc Gel Imaging System (Bio-Rad, CA, USA). When required, membranes were stripped using mild stripping buffer (1.5% glycine, 0.1% SDS, 1% Tween 20, pH 2.2).
6
Colorimetric CA125 Immunoassay Development
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HAuCl4 (hydrogen tetrachloroaurate (III) hydrate), CTAB (hexadecyltrimethylammonium bromide), NaBH4 (sodium borohydride), ascorbic acid, silver nitrate, NHS (N-hydroxysuccinimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), polysorbate 20, paraformaldehyde, silver acetate, hydroquinone, trypsin, trypan blue, anti-rabbit IgG labelled with alkaline phosphatase, 4-nitrophenyl phosphate bis(cyclohexylammonium) salt, milk powder, human serum, human plasma and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay kit as well as all chemicals for the various buffer solutions were purchased from Sigma Aldrich. Monoclonal mouse antibody anti-CA125 (mAb anti-CA125), polyclonal rabbit antibody anti-CA125 (pAb anti-CA125) and CA125 partial recombinant protein were purchased from Novus Biologicals. Alpha-methoxy-omega-mercapto-poly(ethylene glycol) (mPEG-SH) and alpha-carboxy-omega-mercapto-poly(ethylene glycol) (cPEG-SH), Mw ≈ 5000 gmol−1, were provided by Iris Biotech. All cell culture media, fetal calf serum and antibiotics (penicillin and streptomycin) were purchased from Gibco. All chemicals were of analytical grade. Nitrocellulose membranes with pore size of 0.45 μm were purchased from Whatman.
7
HCMV-Specific Antibody Neutralization Assay
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Microplates (96-well μClear high-bind black, Greiner Bio One) were coated with a goat F(ab´)2 anti-human IgG, specific for Fcγ (15 μg/ml, Jackson ImmunoResearch, West Grove, 216 PA, USA) in 100 mM carbonate buffer (pH 9.6) overnight at 4 °C. Non-specific binding was blocked with PBS containing 5% milk powder (Sigma Aldrich) for 2 h at room temperature. The blocking solution was removed and HEK-293 T cells, transiently expressing an HCMV specific human IgG 8 J16, were seeded on the microplate in a serial dilution. In parallel, stably transfected Sp2/0-Ag14 cells secreting a humanized antibody mAb hu2c were seeded serving as unspecific control. Cells were incubated for two days at 37 °C. In order to lyse the cells, the plates were washed with 0.2% Tween 20 in H2O and further three times with MEM5. The reporter virus RV-TB40-BACKL7-SE-EGFP was diluted in MEM5 to 5 × 106 IU/ml (infectious units per ml) and incubated on the plates for 2 h at 37 °C. The unbound virus was removed by three washing steps with MEM5. HFF cells were seeded on the plate in MEM5 with a density of 2.5 × 105 cells per well. After two days of incubation at 37 °C, foci were visualized with an Axio Observer D1 microscope (Zeiss, Oberkochen, Germany). To visualize all footprints in one well, six pictures were taken at a 40-fold magnification and merged.
8
Western Blot Analysis of Membrane Proteins
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Cells were plated in T75 flasks. At 100% confluency, cells were washed and harvested in phosphate buffered saline (PBS). The total cellular membrane proteins were extracted (Plasma Membrane Protein Extraction Kit, abcam, Cambridge, UK) and the concentration was determined by Bradford assay. Three micrograms of each sample were used. Samples were heated at 55 °C for 5 min and separated by SDS-PAGE (Criterion Precast gels, BioRad, Munich, Germany) and transferred to Immuno-Blot PVDF membrane (BioRad). Membranes were blocked in 1× Tris buffered saline (TBS, BioRad) supplemented with 0.2% Tween 20 (Sigma Aldrich) and 5% w/v milk powder (Roth, Karlsruhe, Germany) for 1 h at room temperature. Blots were then incubated with an anti-V5-HRP antibody overnight at 4 °C (Invitrogen). Bound antibodies were detected using an in-house detection solution (100 mM Tris-HCl pH 8.5, 90 mM coumaric acid, 250 mM luminol, 0.04% H2O2).
9
Recombinant Protein Expression Validation
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Western blotting was performed to confirm the expression and purification of the recombinant proteins. The proteins were separated using 12.5% SDS-PAGE and then transferred to nitrocellulose membranes at 100 V for 2 h. The membranes were blocked with 5% milk powder diluted in PBS/0.05% Tween 20 (Sigma) for 2 h and then washed three times with PBS/0.05% Tween 20. After washing, the samples were incubated with His-tag antibodies produced in mice (Sigma-Aldrich®) that had been diluted 2,000× in PBS/BSA 1% for 1.5 h. After three more washes, the membranes were incubated with peroxidase-conjugated rabbit anti-mice-IgG secondary antibodies (Sigma-Aldrich®) that had been diluted 4,000× in PBS/0.05% Tween 20 for 1.5 h. Detection was performed using a Peroxidase Substrate DAB kit (Vector Laboratories®).
10
Immunoblot Analysis of Caspase Activation
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Cell lines (0.1 × 107 cells) were seeded in a 6-well plate (Nunc) and incubated with rfhSP-D (20 µg/ml), together with an untreated control, in a serum-free DMEM-F12 medium. The cells were lysed within the wells using treatment buffer (50 mM Tris-HCl pH 6.8, 2% v/v β-mercaptoethanol, 2% v/v SDS, 0.1% w/v bromophenol blue, and 10% v/v glycerol) and transferred to pre-cooled microcentrifuge tubes followed by sonication for 15 s. The samples were heated at 100°C for 10 min and subjected to SDS-PAGE (12% w/v) for 90 min at 120 V. The SDS-PAGE separated proteins were then electrophoretically transferred onto a nitrocellulose membrane (Thermo Fisher) using an iBLOT (Thermo Fisher). The membrane was then blocked using 5% w/v dried milk powder (Sigma) in 100 ml PBS for 2 h on a rotatory shaker at room temperature. The membrane was incubated with rabbit anti-human caspase primary antibodies (anti-cleaved caspase 3; anti-cleaved caspase 8; Cell Signaling) at 4°C overnight, followed by incubation with secondary Goat anti-rabbit IgG HRP-conjugate (1:1,000; Promega) for 1 h at room temperature. The membrane was washed with PBST (PBS + 0.05% Tween 20) three times, 10 min each time. The color was developed using 3,3′-diaminobenzidine substrate kit (Thermo Fisher).
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