Differentiated SGBS adipocytes and undifferentiated control cells were washed once with PBS, and then suspended in lysis buffer which consists of 50 nM Tris-HCl, 0,1% Triton X-100 (Sigma), 15 mM 2-mercaptoethanol (Sigma), 1 mM EDTA (Sigma) and protease inhibitor (Sigma). The lysates were suspended in 5x Laemmli loading buffer and boiled for 10 min at 100 °C. Equal amount of proteins were loaded onto a 12%-SDS-polyacrylamide gel, and transferred onto a PVDF Immobilon-P Transfer Membrane (Merck-Millipore, Germany). Then the membranes were blocked in 5% skimmed milk (Sigma) for 1 hour. For overnight, membranes were probed at 4 °C with primary antibodies: monoclonal anti-UCP1 (1:1000, R&D Systems, MAB6158), polyclonal anti-UCP1 (1:500, Sigma, U6382; 1:500, Thermo Scientific, PA1-24894), anti-OXPHOS (1:1000, Abcam, UK, ab110411), and anti-actin (1:10000, Sigma, A2066), in TBS-T containing 1% non-fat skimmed milk, followed by the incubation with horseradish-peroxidase-conjugated species corresponding secondary antibodies (Sigma) for 1 hour at room temperature. Immunoreactive proteins were visualized by Immobilion western chemiluminescence substrate (Merck-Millipore). ImageJ software was used to carry out the densitometry measurements.
Skimmed milk
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Skimmed milk is a type of dairy product that has had most of its fat content removed. It is a common ingredient used in various laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (9 mentions)
Lumi light, by Roche (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Image studio lite, by LI COR (4 mentions)
Immobilon p, by Merck Group (4 mentions)
Quantity one chemidoc xrs software, by Bio-Rad (4 mentions)
Clarity western ecl substrate, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Pvdf membrane, by Cytiva (3 mentions)
Nitrocellulose membrane, by LI COR (3 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (3 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (3 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail tablet, by Roche (3 mentions)
P1379, by Merck Group (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Trans blot semidry transfer unit, by Bio-Rad (3 mentions)
Odyssey infrared imager, by LI COR (3 mentions)
Super block dry blend, by Thermo Fisher Scientific (3 mentions)
86 protocols using skimmed milk
1
Western Blot Analysis of Adipocyte Markers
2
Western Blot Analysis of CCND1 Expression
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Total cell lysates were prepared and analyzed by 10% SDS-PAGE as previously described (30 (link),32 (link),33 (link)). Membranes were blocked with 5% (w/v) skimmed milk (Merck KGaA, Darmstadt, Germany) for 1 h at room temperature and incubated with the following primary antibodies overnight at 4°C: Anti-CCND1 (cat. no., 2922; dilution, 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-β-actin (cat. no., GTX26276; dilution, 1:5,000; GeneTex, Inc., Irvine, CA, USA). Membranes were then incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit IgG (cat. no. 7074S; 1:3,000; Cell Signaling Technology, Inc.) or peroxidase-conjugated sheep anti-mouse IgG antibody (ECL anti-mouse IgG; cat. no. NA931V; 1:3,000) (Amersham Pharmacia Biosciences, Buckinghamshire, U.K.). Immunodetection was performed using the horseradish peroxidase-based SuperSignal Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). For quantification, the bands were measured with the AlphaImager 2200 Imaging System (Alpha Innotech; Bio-Techne, Minneapolis, MN, USA), and the densities of the CCND1 bands were normalized to those of the β-actin bands. CCND1 expression was quantified and described as a ratio to β-actin expression (CCND1/β-actin ratio).
3
Protein Extraction and Western Blot Analysis
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Cell pellets were lysed in radio‐immunoprecipitation assay buffer [50 mm Tris/HCl (pH 8), 150 mm NaCl, 1% Igepal CA‐630, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mm sodium orthovanadate and protease inhibitor cocktails], for 30 min on ice and centrifuged for another 30 min at maximum speed at 4 °C. Protein concentration was assessed by Bradford assay (Quick Start™ Bradford 1× Dye Reagent; Bio‐Rad Laboratories, Hercules, CA, USA). After gel electrophoresis, proteins were transferred onto the PVDF or nitrocellulose membrane by means of Trans‐Blot® Turbo™ Transfer System (Bio‐Rad Laboratories) and blocked by 1‐h incubation in 5% skimmed milk (Merck Group, Darmstadt, Germany) in PBST at room temperature. The following antibodies were used in immunoblotting: anti‐H3 (ab1791; Abcam, Cambridge, UK; RRID:AB_302613), anti‐H3AcK9 (ab10812; Abcam; RRID:AB_297491), anti‐H3AcK14 (#7627; Cell Signaling, Danvers, MA, USA; RRID:AB_10839410) and anti‐β‐Actin (#3700; Cell Signaling; RRID:AB_2242334), all overnight at 4 °C. Next, membranes were washed and treated with secondary antibody for 1 h at RT. Chemiluminescent signals were detected by LI‐COR C‐Digit (LI‐COR Biosciences, Lincoln, NE, USA), and the data were quantified using imagej software (National Institutes of Health, Bethesda, MD, USA) (RRID:SCR_003070).
4
Western Blot Analysis of PLCζ and PAWP in Globozoospermia
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Briefly, approximately 35 µg of protein was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (PVDF, Biorad, USA). The membranes were blocked with skimmed milk (Merck, USA) and polyclonal antiPLCζ antibody (1:32000, Covalab, France), polyclonal anti-PAWP antibody (1:5000, abcam, UK) and monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), clone 6C5 (1:5000, Millipore, USA), were used as specific primary antibodies. After three times washing, the secondary antibodies, used for PAWP and PLCζ, were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and for GAPDH was anti-rabbit IgG (all purchased from Dako, Japan). After three times washing, target proteins band were detected with an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, Germany). The fire reader (Uvitec, UK) was used for recording chemiluminescence images. Densitometric analysis of the images was performed by Quantity One Software v 4.6.9 (Bio-Rad, Germany). Results were expressed as mean relative intensity (mean intensity of the patient’s band/mean intensity of fertile bands) (17 (link)). Figure 1 showed Western blot of PLCζ and PAWP in infertile men with globozoospermia (n=4) and fertile men (n=5).
5
Western Blot Analysis of Histone Modifications
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Cell pellets were lysed in radioimmunoprecipitation assay buffer (50 mM tris-HCl (pH 8), 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mM sodium orthovanadate, and protease inhibitor cocktails), for 30 min on ice and centrifuged for another 30 min at maximum speed at 4 °C. Protein concentration was assessed by Bradford assay (B6916, Quick Start™ Bradford 1× Dye Reagent, Bio-Rad Laboratories, Hercules, CA, USA). After gel electrophoresis, proteins were transferred onto the PVDF or nitrocellulose membrane by means of Trans-Blot® Turbo™ Transfer System (1704150, Bio-Rad Laboratories) and blocked by 1-h incubation in 5% skimmed milk (70166, Merck Group, Darmstadt, Germany) in PBST at room temperature. The following antibodies were used in immunoblotting: anti-Histone H3 (ab1791, Abcam; 1:1000), anti-H3AcK9 (ab10812, Abcam; 1:1000), anti-H3AcK14 (#7627, Cell Signaling, Danvers, MA, USA; 1:1000), anti-β-Actin (#3700, Cell Signaling; 1:1000), anti-CIITA (TA319682, Origene, Rockville, MD, USA; 1:1000) all overnight at 4 °C. Next, membranes were washed and treated with secondary antibody for 1 h at RT. Chemiluminescent signals were detected by LICOR C-Digit (LI-COR Biosciences, Lincoln, NE, USA).
6
Western Blot Analysis of Extracellular Vesicles
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Samples were mixed with sample reducing agent (Invitrogen, Thermo Fischer Scientific) and LDS sample buffer (Invitrogen, Thermo Fischer Scientific) and heated to 95°C for 10 min before separation by SDS‐polyacrylamide gel electrophoresis (BIO‐RAD). Proteins were then transferred to a PVDF membrane (Merck) and activated in 99% Ethanol. After blotting, the membrane was blocked for 30 min in 5% skimmed milk (Merck) and incubated at 4°C overnight with primary antibody (Table 1 ). The membrane was washed in Tris‐Buffered saline with Tween‐20 (TBST, 20 mM Tris‐Base, 137 mM NaCl, 0.05% Tween‐20 [Merck], pH 7.6) and incubated for 1 h at room temperature with secondary antibody (Table 1 ). Membranes were developed with ECL plus and imaged by a Molecular Imager (ChemiDoc XRS +, BIO‐RAD) using Image Lab software (BIO‐RAD). The EV band densities were normalized to cellular protein concentration used as an estimate of cell abundance.
7
Screening of Microbial Enzymatic Activities
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All chemicals and growth media were purchased from commercial sources. LB broth and LB agar medium were bought from A&A Biotechnology (Gdynia; Poland). Mannitol salt phenol−red agar, Spirit blue agar, tributyrin agar, skimmed milk, starch, carboxymethylcellulose, guaiacol, X−gal (5−bromo−4−chloro−3−indolyl−β−D−galactopyranoside), Tween 80, cottonseed oil, Lugol’s solution, Congo red dye, and PBS tablets (pH 7.4) were purchased from Merck (Darmstadt, Germany). Ultrapure H2O (18.0 MΩ) was produced with the Milli−Q Advantage A10 system (Millipore, Billerica, MA, USA).
8
ELISA for CCHFV IgG Antibody Detection
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CCHFV IgG antibodies were detected using ELISA assay. For this purpose, Nunc MaxiSorp™ ELISA plates (Thermo Fisher Scientific, USA) were coated with diluted mouse hyperimmune ascetic fluid (1:1000) in phosphate-buffered saline (PBS 1X) and incubated overnight at 4°C. After washing, CCHFV antigen in PBS (1:500 dilution), containing 0.5% Tween (Merck, Germany) (PBST) and 3% skimmed milk (Merck, Germany) (PBSTM) was added and then incubated for three hours at 37°C. After adding the diluted sera in PBSTM (1:100) and incubation for one hour at 37°C, peroxidase-labeled anti-ovine/caprineIgG (ICN Biomedicals Inc., USA; 1:700 dilution in PBSTM) was added and incubated again for one hour at 37°C. After the washing step (three times), hydrogen peroxide (H 2 O 2 ) (MP Biomedical, France) and 3,3', 5,5'-tetramethylbenzidine (TMB; MP Biomedical, France) were added and incubated for 15 minutes at room temperature. Finally, the reaction was terminated using H 2 SO 4 (Sigma, USA), and the plates were read at 450 nm.
9
Phage ELISA for AGS-specific Binders
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The enrichment of AGS-specific binders was verified by polyclonal phage ELISA.27 (link) The amplified phages after each selection round were tested for binding to AGS and MKN-45 cells. The cells were detached as previously described and blocked in 2% (w/v) skimmed milk (Merck, Kenilworth, NJ, USA) in PBS and distributed approximately 5x105 (link) cells/well in a 96-well flat-bottomed tissue culture plate (SPL Life Sciences, Gyeonggi-do, South Korea).28 (link) The blocked phages were allowed to bind to the cells for 90 min at 4°C. Followed by several washing steps, the cell surface-binding phage antibodies were detected with 1:5000 dilution of murine anti-M13 monoclonal antibody (GE Healthcare, Little Chalfont, UK) and HRP-conjugated goat anti-mouse IgG (Invitrogen, Waltham, Massachusetts, USA).29 (link) The substrate solution, TMB (3,3,5,50-tetramethylbenzidine), was added and incubated in the dark for 30 min. The cells were spun down at maximum rate and 100 µL/well of the supernatant was transferred to a new plate which had already been filled with 50 µL of 1 M sulfuric acid per well to stop the color development.30 (link) The absorbance was read at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
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The enrichment of AGS-specific binders was verified by polyclonal phage ELISA.27 (link) The amplified phages after each selection round were tested for binding to AGS and MKN-45 cells. The cells were detached as previously described and blocked in 2% (w/v) skimmed milk (Merck, Kenilworth, NJ, USA) in PBS and distributed approximately 5x105 (link) cells/well in a 96-well flat-bottomed tissue culture plate (SPL Life Sciences, Gyeonggi-do, South Korea).28 (link) The blocked phages were allowed to bind to the cells for 90 min at 4°C. Followed by several washing steps, the cell surface-binding phage antibodies were detected with 1:5000 dilution of murine anti-M13 monoclonal antibody (GE Healthcare, Little Chalfont, UK) and HRP-conjugated goat anti-mouse IgG (Invitrogen, Waltham, Massachusetts, USA).29 (link) The substrate solution, TMB (3,3,5,50-tetramethylbenzidine), was added and incubated in the dark for 30 min. The cells were spun down at maximum rate and 100 µL/well of the supernatant was transferred to a new plate which had already been filled with 50 µL of 1 M sulfuric acid per well to stop the color development.30 (link) The absorbance was read at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
10
Qualitative Assessment of LAB Proteolytic Activity
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The proteolytic activity of the LAB was qualitatively detected after overnight growth in optimal medium or RSM, using the skimmed milk agar as described by Raveschot et al. (2020) with slight modifications. A 50 mL of LAB biomass after centrifugation of 50 μL of cell supernatant was placed on agar containing 1 % (m/V) skimmed milk (MerckKGaA, Darmstadt, Germany) and incubated at 30 °C for 48 h. Proteolytic activity was indicated by the occurrence of a clear hydrolysis zone. All analyses were performed in triplicates.
Selected strains, that showed caseinolytic activity, were inoculated with the vertical line test on skimmed milk with 1 % (m/V) agar. After 48 h of incubation, transparent zones around the bacterial growth were photographed.
Selected strains, that showed caseinolytic activity, were inoculated with the vertical line test on skimmed milk with 1 % (m/V) agar. After 48 h of incubation, transparent zones around the bacterial growth were photographed.
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