Proteins were extracted from cells or lung tissue using RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% PMSF with 1% phosphatase inhibitor (both obtained from Beyotime) according to the protocol provided by the reagent manufacturer. Protein concentrations were determined using the BCA protein assay Kit (Boster Biological Technology, Wuhan, China). Loading Buffer (LT101S, Epizyme, Shanghai, China) was added to the protein extraction solution, heated to 95 °C for 5 min, and stored at low temperatures. Proteins were transferred to PVDF membranes (Merck, MA, USA) for 2 h. Subsequently, the gels were closed for 2 h using 5% skim milk powder (BD, Franklin Lakes, NJ, USA) (dissolved in tris-buffered saline and supplemented with 0.1% tween-20 (TBST)). PVDF membranes were incubated overnight at 4° with primary antibodies against HIF-1α, SMAD 3, PSMAD 3, β-actin, COL1α1, and α-SMA genes. PVDF membrane was washed three times with 1× TBST and was incubated with secondary antibody at 37° for 2 h and then rewashed three times with 1× TBST. Proteins were detected using ECL detection reagent (Beyotime) and fluorescent signals were collected using Amersham Imager 600 system (GE, Fairfield, UK). To confirm that the sample sizes were equal, we used β-actin to detect the blots and analyzed them using ImageJ software.
Skim milk powder
Manufactured by BD
Sourced in United States
Skim milk powder is a dried dairy product made by removing the fat and water content from fresh milk. It serves as a convenient and shelf-stable ingredient for various food and beverage applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (2 mentions)
Amersham imager 600 system, by GE Healthcare (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Ecl detection reagent, by Beyotime (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Gapdh, by Abcam (1 mentions)
Phenylmethylsulfonyl fluoride, by Thermo Fisher Scientific (1 mentions)
Ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Thiourea, by Thermo Fisher Scientific (1 mentions)
Ammonium sulphate, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Trypsin, by BD (1 mentions)
Gelatin, by BD (1 mentions)
Analytical grade methanol, by Thermo Fisher Scientific (1 mentions)
Lc ms grade water, by Thermo Fisher Scientific (1 mentions)
Tryptone, by BD (1 mentions)
14 protocols using skim milk powder
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Osteogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed to detect the expression levels of osteogenic differentiation, autophagy and NF-κB signalling pathway marker proteins. Briefly, total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China), and the BCA method was used to detect the protein concentration. The loading volume was determined with 20 μg as the loading amount. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared to separate proteins, which were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the membrane was blocked with 5% skim milk powder (BD, Franklin Lakes, NJ, USA) at room temperature for 2 h and incubated with P21, ALP, RUNX2, Atg5, LC3, p-IκB, IκB, p-P65, P65 and glyceraldheyde 3-phosphate dehydrogenase (GAPDH) antibodies (Abcam, Cambridge, UK; dilution 1:200) overnight at 4°C. The second day after washing the membrane, the corresponding secondary antibody (Abcam; dilution 1:1000) was incubated at room temperature for 1 h. A chemiluminescence imaging system (Tanon, Shanghai, China) was used for development, and the results were analysed using ImageJ software for greyscale analysis.
3
Potato Protein Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plant materials, bacterial strains, and chemicals.
Solanum tuberosum DM1 (sometimes known as DM1-3) and S. chacoense M6 were grown in a greenhouse at 18 to 24°C with a day length of 16 h. Plants were grown in ProMix BX general purpose mix and fertilized with Osmocote Plus 15-9-12 (Scotts-MiracleGro). Tubers were used for protein extraction. NaCl, ethylenediaminetetraacetic acid (EDTA), thiourea, dithiothreitol (DTT), phenylmethylsulfonyl fluoride, Tris-HCl, ammonium sulphate, polyvinylpolypyrrolidone (PVP), and hydrochloric acid were purchased from Fisher Chemicals (Thermo Fisher Scientific). Pb1692 was used for all experiments in this study (Duarte et al. 2004 (link)). Nutrient broth (NB), agar, gelatin, skim milk powder, trypsin, and cPI were purchased from Difco Laboratories (Thermo Fisher Scientific).
Solanum tuberosum DM1 (sometimes known as DM1-3) and S. chacoense M6 were grown in a greenhouse at 18 to 24°C with a day length of 16 h. Plants were grown in ProMix BX general purpose mix and fertilized with Osmocote Plus 15-9-12 (Scotts-MiracleGro). Tubers were used for protein extraction. NaCl, ethylenediaminetetraacetic acid (EDTA), thiourea, dithiothreitol (DTT), phenylmethylsulfonyl fluoride, Tris-HCl, ammonium sulphate, polyvinylpolypyrrolidone (PVP), and hydrochloric acid were purchased from Fisher Chemicals (Thermo Fisher Scientific). Pb1692 was used for all experiments in this study (Duarte et al. 2004 (link)). Nutrient broth (NB), agar, gelatin, skim milk powder, trypsin, and cPI were purchased from Difco Laboratories (Thermo Fisher Scientific).
4
Potato Growth and Metabolite Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plant materials, bacterial strains, and chemicals.
S. tuberosum (DM1) and S. chacoense (Hassl.) (M6) were grown in a greenhouse at 18 to 24°C with a 16-h day length. Plants were grown in ProMix Bx General Purpose mix, fertilized with Osmocote Plus 15-9-12 (Scotts-MiracleGro, U.S.A.), and irrigated to saturation every other day until used for assays. Aphids and other pests were managed with Botaniguard ES and Molt-X (BioWorks, U.S.A.), Entrust SC (Corteva, U.S.A.), Distance IGR (Valent Biosicences, U.S.A.), Judo and Azatin (OHP Inc., U.S.A.), Avid 0.15EC (Syngenta, U.S.A.), and Compass (Bayer, U.S.A.)
Tubers and 1-month old stems were used for extraction of metabolites. LC-MS-grade water, analytical-grade methanol, ACN, and hydrochloric acid were purchased from Fisher Chemicals (Thermo Fisher Scientific, U.S.A.). P. brasiliense Pb1692 was used for all experiments in this study (Duarte et al. 2004) . Nutrient broth (NB), agar, skim milk powder, tryptone, Congo red, and NaCl were purchased from Difco Laboratories (Thermo Fisher Scientific). Bacteria were grown at 30°C under continuous shaking conditions.
S. tuberosum (DM1) and S. chacoense (Hassl.) (M6) were grown in a greenhouse at 18 to 24°C with a 16-h day length. Plants were grown in ProMix Bx General Purpose mix, fertilized with Osmocote Plus 15-9-12 (Scotts-MiracleGro, U.S.A.), and irrigated to saturation every other day until used for assays. Aphids and other pests were managed with Botaniguard ES and Molt-X (BioWorks, U.S.A.), Entrust SC (Corteva, U.S.A.), Distance IGR (Valent Biosicences, U.S.A.), Judo and Azatin (OHP Inc., U.S.A.), Avid 0.15EC (Syngenta, U.S.A.), and Compass (Bayer, U.S.A.)
Tubers and 1-month old stems were used for extraction of metabolites. LC-MS-grade water, analytical-grade methanol, ACN, and hydrochloric acid were purchased from Fisher Chemicals (Thermo Fisher Scientific, U.S.A.). P. brasiliense Pb1692 was used for all experiments in this study (Duarte et al. 2004) . Nutrient broth (NB), agar, skim milk powder, tryptone, Congo red, and NaCl were purchased from Difco Laboratories (Thermo Fisher Scientific). Bacteria were grown at 30°C under continuous shaking conditions.
5
Evaluation of Proteolytic and Lipolytic Activities
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pH values were measured on the mini-cheeses that were mixed with a spatula. Proteolytic activity was determined on calcium caseinate agar modified according to Frazier and Rupp (Merck, Darmstadt, Germany) supplemented with 1% (w/v) skim milk powder (BD DifcoTM). Lipolytic activity was determined on tributyrin agar (Sigma-Aldrich) supplemented with 1% (w/v) tributyrin (Sigma-Aldrich). Ten microliters of cell suspensions at 106 CFU/ml in physiological water (NaCl 9 g/l) were spot-inoculated onto the plates, which were then incubated at 15 or 25°C for 21 days. Proteolytic and lipolytic activities were evaluated by the formation of a clear zone around the spots.
6
Irbesartan and Atorvastatin Effects on Lipid Profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Irbesartan was purchased from Jiangsu Hengrui Medicine Co., Ltd. (state approval no. H20000513; Lianyungang, China). Atorvastatin calcium was purchased from Beijing Jialin Pharmaceutical Co., Ltd. (state approval no. H20093819; Beijing, China). TRIzol and RT-PCR kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) kits (all from Cyttel Bioscience Inc., Beijing, China). Skim milk powder (BD Biosciences, San Jose, CA, USA), rabbit anti-rat sPLA2-V polyclonal antibody (cat no. 16009-1-AP; 1:1,000) and goat anti-rabbit-HRP secondary polyclonal antibody (cat. no. SA00001-2; 1:800) were purchased from Wuhan Sanying Biotechnology, Wuhan, China. Protein electrophoresis buffer, transfer membrane buffer and washing, all from Sangon Biotech (Shanghai) Co., Ltd.
7
Gene Expression Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol (Life Technologies, New York, NY, USA); chloroform and isopropyl alcohol (Beijing Chemical Co., Ltd., Beijing, China); M-MLV reverse transcriptase; DNase I (both from Life Technologies); SYBR® Premix Ex Taq™ II (Takara Bio Inc., Liaoning, China); DNA Marker (TransGen Biotech, Beijing, China); primer synthesis (Beijing Genomics Institute, Guangdong, China); RIPA protein lysate (Solarbio, Beijing, China); actin and STK33 rabbit anti-human primary antibodies (Cell Signaling Technology, Boston, MA, USA); BCA kit (Life Technologies); skim milk powder (BD Biosciences, New Jersey, NY, USA); horseradish peroxidase labeled goat anti-rabbit IgG secondary antibody (Cell Signaling Technology); NC membrane (Millipore, Billerica, MA, USA); luminescent substrate kit (TransGen Biotech); SP kit (Cell Signaling Technology).
8
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed on ice for 30 minutes in RIPA lysis buffer (Meilunbio, China) containing phenylmethanesulfonyl fluoride (0.1 mg/mL). The supernatant was centrifuged to obtain the sample of the protein. The protein was quantified by a BCA kit (Meilunbio, China) with 10 μL of the supernatant taken. 40μg protein sample was loaded to 8% -12% gel electrophoresis SDS-PAGE. The protein was transferred to the polyvinylidene fluoride (PVDF) Membrane (Millipore, USA, ThermoFisher, USA). The PVDF membrane was blocked in a blocking solution by the TBST buffer (Meilunbio, China), which contains 5% skim milk powder (BD, USA) for one hour. The PVDF membrane was incubated with the primary antibody at 4 °C overnight and then incubated with the corresponding secondary antibody for 1 hour at room temperature. After being washed three times with TBST buffer (Meilunbio, China), The PVDF membrane was detected by Western fluorescence assay Beyo ECL Plus (Beyotime, China, Meilunbio, China). The following antibodies were used: Anti-CD44 (1:2000, CST, USA), Anti-EpCAM (1:1000, CST, USA), Anti-MKL-1 (1:1000, CST, USA), Anti-GAPDH (1:1000, Abcam, USA).
9
SARS-CoV-2 Concentration in Moore Swabs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Skim milk workflow (Figure 1 ) was used for SARS-CoV-2 concentration in Moore swab samples described by Liu et al. (2022 (link)) because these samples had higher turbidity. In brief, a 5% (w/v) skim milk solution was prepared by dissolving 5 g of skim milk powder (BD, #232100, Sparks, MD) in 100 ml of distilled water. Before the flocculation step, the liquid sample was squeezed from a Moore swab, processed using BigMixer 400 CC (Interscience for Microbiology, France) at speed 2 for 30 s, and was adjusted to 3.5 using 6 N HCL. Then, skim milk was added to the final concentration of 1% in 250 ml of wastewater, followed by the addition of 105 GC of BRSV and shaking for 2 h. After this step, the sample was centrifuged (12,000 × g for 30 min), followed by RNA extraction using the Applied Biosystems MagMaxTM nucleic acid isolation kit (Thermo Fisher Scientific #48310). Finally, 60 μl of RNA was achieved from the extraction procedure.
10
Arabidopsis Nuclear Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear proteins were extracted from 2-week-old Arabidopsis seedlings according to a previously published method [77 (link)]. The nuclear proteins were separated on SDS-PAGE gels and transferred to 0.45-μm nitrocellulose membranes (Amersham). The membranes were blocked with 5% skim milk powder (BD) in 50 mM Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) at room temperature for 1 hour and subsequently incubated with anti-H3 (Abcam, ab1791) (diluted at 1:3,000 in TBS-T with 5% skim milk powder), anti-H3K27me3 (Millipore, 07–449) (diluted at 1:500) [78 (link),79 (link)], or anti-GFP (EASYBIO, BE2002) (diluted at 1:1,000) at room temperature for 1 hour. The membranes were washed three times with TBS-T, then incubated with goat anti-rabbit IgG-HRP antibody (EASYBIO, BE0101) (diluted at 1:10,000) for 1 hour at room temperature. Finally, the membranes were washed three times with TBS-T, and the protein bands were detected using a chemiluminescent system (Thermo, 34075).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!