Total protein in PC cells was extracted using radioimmunoassay precipitation lysis buffer (Beyotime Biotechnology, Suzhou, China) containing phenylmethylsulfonyl fluoride (Servicebio, Wuhan, China). Protein concentrations in the samples were determined using the bicinchoninic acid method (Servicebio, Wuhan, China). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gels (Meilune, Dalian, China)and thereafter, transferred to polyvinylidene fluoride membranes (Thermo Scientific, USA). The membranes were blocked with skim milk powder (Beyotime Biotechnology, Suzhou, China) and incubated with primary antibodies (GADD45A [1:500], Cat No. A1797, Abconal, China; CDK1 [1:500], Cat No. 19532-1-AP, Proteintech, China; CCNB1 [1:500], Cat No. 28603-1-AP, Proteintech, China; N-cadherin [1:500], Cat No. 22018-1-AP, Proteintech, China; E-cadherin [1:500], Cat No. 20874-1-AP, Proteintech, China; and β-actin [ACTB], 1:500; Cat No. 20536-1-AP, Proteintech) for 16 hours at 4℃. After washing twice with Tris-buffered saline containing 0.1% Tween‐20, the membranes were incubated with secondary antibody and visualized using an enhanced chemiluminescence reagent. ACTB was used as the loading control to calculate the relative protein expression.
Skim milk powder
Manufactured by Beyotime
Sourced in China
Skim milk powder is a dehydrated form of nonfat milk. It is produced by removing the fat and water content from fresh milk, leaving behind a fine, powdered substance. Skim milk powder is a versatile ingredient used in various food and beverage applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Phenylmethylsulfonyl fluoride, by Wuhan Servicebio Technology (1 mentions)
Ccnb1, by Proteintech (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Ecl chemiluminescence, by Beyotime (1 mentions)
Gapdh, by Wuhan Servicebio Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Yeasen (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Sds page, by GenScript (1 mentions)
Anti β actin antibody, by Beyotime (1 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
β actin, by Wuhan Sanying Biotechnology (1 mentions)
Cell protein extraction kit, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
11 protocols using skim milk powder
1
Quantitative Protein Analysis in PC Cells
2
Evaluating Genotoxicity of Nano-Conjugated Gefitinib
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A549 and H1299 cells were cultured in six-well plates with a seeding density of 1 × 106 cells/well for 24 h. Subsequently, the cells were treated with 50 μM of GEF, GEF@NCA, or GEF@TSBO for an additional 24 h. After treatment, the cells were harvested and lysed using protease inhibitors on ice for 30 min. The lysate was then centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was quantified with a BCA Protein Assay Kit (Yeasen Bio., Shanghai, China). Proteins were separated on a 10% Bis-Tris polyacrylamide gel (Beyotime, Nantong, China) and then transferred onto a PVDF membrane (BioRad, Hercules, CA, USA). The membrane was blocked with 5% skim milk powder (Beyotime, Nantong, China) and incubated with primary antibodies against GAPDH (Servicebio, Wuhan, China), γ-H2AX (CST, Danvers, MA, USA), and PARP (CST, USA) overnight. After an hour of incubation, protein expression levels were detected using ECL chemiluminescence (Beyotime, Nantong, China) with a suitable secondary antibody (Yeasen Biotechnology, Shanghai, China).
3
Htra2 Protein Expression Analysis
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Htra2 expression was assessed by western blot analysis. Cochleae from 4-week-old mice were collected and lysed in RIPA buffer (Beyotime Biotechnology, Haimen, China) supplemented with phenylmethanesulfonyl fluoride (Beyotime Biotechnology). Proteins were separated by SDS-PAGE (GenScript, Piscataway, NJ) and transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA). The membranes were blocked in 5% skim milk powder (Beyotime Biotechnology) and probed with anti-Htra2 (Cell Signaling Technology, Danvers, MA) or anti-β-actin antibodies (Beyotime Biotechnology) at 4°C overnight. The membranes were labeled with HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime Biotechnology) for 1 h at room temperature, and the bands were visualized using western Blot ECL Blotting Substrate (Bio-Rad, Hercules, CA). The images were analyzed through ImageJ software.
4
Synthesis and Characterization of HBA-PEG Conjugate
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p-Hydroxybenzyl alcohol (HBA), oxalyl chloride (OC), RAPA, and poly-(ethylene glycol)2000 (PEG2000) were obtained from Shanghai Aladdin Bio-Chem Technology Co, Ltd. (Shanghai, China). Tetrahydrofuran (THF), hydrogen peroxide (H2O2, 30%), DMSO, deuterium DMSO, and trichloromethane were purchased from Chongqing Chuandong chemical (group) Co, Ltd. (Chongqing, China). Polyclonal antibodies of CXCR4, CD47, and β-actin were acquired from Wuhan Sanying Biotechnology Co, Ltd. Proteintech Group. inc. (Wuhan, Hubei, China). Coomassie bright blue stain, BSA protein concentration tester, skim milk powder, TBST, cell protein extraction kit, DHE, DAPI, DiO, and DiD were purchased from Shanghai Beyotime biotechnology Co, Ltd. (Shanghai, China). SDF-1, ox-LDL, and AMD3100 were obtained from Beijing Solarbio Technology Co, Ltd. (Beijing, China). TTC and EB were acquired from Nanjing Jiancheng Bioengineering Institute. (Nanjing, Jiangsu, China). and Shanghai Yuanye Biotechnology Co, Ltd. (Shanghai, China), respectively.
5
Protein Expression Analysis in HCC Cells
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HCC cells were lysed using radioimmunoprecipitation assay buffer (Beyotime, Suzhou, China) containing 1% phenylmethylsulfonyl fluoride (Beyotime). Total proteins in HCC cells were extracted by centrifugation at 4°C and protein concentrations were determined using the BCA method. A total of 30 μg of protein from each sample was electrophoresed in sodium dodecyl sulfate-polyacrylamide gels (Meilune, Dalian, China) and then transferred to polyvinylidene fluoride membranes (Thermo Scientific, USA). Membranes were blocked using TBST containing 5% skim milk powder (Beyotime, Suzhou, China). Primary antibodies against CFHR3 (1:500; Cat No. 16583-1-AP; Proteintech, Wuhan, China), EGLN3 (1:500; Cat No. 18325-1-AP; Proteintech, Wuhan, China), CHGA (1:500; Cat No. 10529-1-AP; Proteintech, Wuhan, China) and actin beta (ACTB) (1:1000; Cat No. 20536-1-AP; Proteintech) were added and incubated overnight at 4°C. After washing three times with PBS, the membranes were incubated with secondary antibodies for 2 h and visualized using ECL reagents (Boster, Wuhan, China). The relative expression of CFHR3, EGLN3 and CHGA was normalized to that of ACTB.
6
Detailed Antibody Analysis Protocol
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Detailed information on antibodies was listed in Supplementary Table S4 . Phosphate buffered saline (PBS), Tween‐20, RIPA lysis buffer, phenylmethylsulfonyl fluoride (PMSF), phosphotungstic acid negative stain solution (2%), and glutaraldehyde (2.5%, EM Grade) were obtained from Solarbio (Beijing, China). Methanol was obtained from Concord Technology (Tianjin, China). Sodium dodecyl sulfate (SDS), BCA Protein Assay Kit, SDS‐PAGE sample loading buffer, prestained colour protein marker, nitrocellulose membrane, polyvinylidene difluoride membrane, skim milk powder, and BeyoECL Plus were obtained from Beyotime Biotechnology (Shanghai, China). Precast Protein Improve Gels (4%–20%, 10 wells) were obtained from Yeasen (Shanghai, China). The coating buffer was obtained from BioLegend (California, USA). Casein blocking buffer (1%) was obtained from Leagene Biotechnology (Beijing, China). Tris(hydroxymethyl) aminoethane, 2‐[4‐(2‐hydroxyethyl)‐1‐piperazinyl]ethanesulfonic acid (HEPES), ethylene diamine tetraacetic acid (EDTA), and glycine was obtained from Aladdin (Shanghai, China). TMB chemiluminescence chromogenic solution and stop solution were obtained from Abcam (Cambridge, MA).
7
Western Blot Analysis of TREM1 Expression
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Treated cell samples were lysed 30 min in RIPA buffer (Thermo Fisher Scientific) supplemented with the protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Beyotime Biotechnology, Shanghai, China). Protein lysates were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (0.22 μm, Merck Millipore; Darmstadt, Germany). Membranes were blocked at room temperature for 1 h in Tris-buffered saline with Tween-20 (TBST;10 mM Tris, 150 mM NaCl, and 0.1% Tween 20) containing 5% skim milk powder (Beyotime) and incubated overnight with primary antibody at 4°C, followed the next day by incubation with a secondary antibody conjugated to horseradish peroxidase (HRP) reconstituted in antibody dilution buffer (dilution 1: 5000; Beyotime) for 1 h at room temperature. Specific proteins were visualized with enhanced chemiluminescence (ECL, Millipore; Bedford, MA, USA) according to the manufacturer's protocol. The following primary antibodies were used: rabbit anti-TREM1 (PA5-95477, Thermo Fisher Scientific); rabbit anti-ACTB (20536-1-AP, Proteintech Group, Inc.; Wuhan, China).
8
Western Blot Analysis in HeLa Cells
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48 h after the transfection of HeLa cells, the culture medium was removed. After PBS rinsing, the cells were lysed with protein lysate (Beyotime, China), and the total proteins were collected. Then the protein concentration was detected by BCA (Beyotime, China) protein quantitative kit. After adding proper amount of SDS loading buffer (Beyotime, China), denaturation was carried out in boiling water at 100°C for 5 min. Then 12% SDS-PAGE electrophoresis (Beyotime, China) was performed. Protein bands were transferred to PVDF membranes (Beyotime, China) by western transmembrane system. Subsequently, the PVDF membrane was sealed in 5% skim milk powder (Beyotime, China) and incubated for 4 h. After washing with TBST (Beyotime, China), the PVDF membrane was incubated with primary antibody at 4°C overnight. After washing, the membrane was incubated with the secondary antibody at room temperature for 60 min. Finally, the PVDF membrane was stained with western TMB substrate (Beyotime, China; P0211) and scanned. The antibodies used were β-actin (Beyotime, China; AF5003), TYMS (ABCAm, United States, ab108995) and horseradish peroxidase labeled goat anti-rabbit IgG (Beyotime, China; A0208).
9
Protein Expression Analysis Protocol
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Cells were lysed with RIPA buffer (Beyotime, China) to obtain total protein, and protein concentration was determined by BCA method. An equal amount of the sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the protein was transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, USA). After blocking with 5% skim milk powder (Beyotime, China) at room temperature for 2 h, the membrane and primary antibody were incubated overnight at 4 °C. Primary antibodies: CCDC170 (coiled-coil domain-containing 170) (#bs-15255R, Bioss, Beijing, China), COL14A1 (collagen type XIV alpha 1 chain) (#AF0573, Affinity Biosciences, Cincinnati, OH, USA) and THBS2 (thrombospondin 2) (#bs-7524R, Bioss, Beijing, China). Then it was incubated with horseradish peroxidase secondary antibody (Beyotime, China) at room temperature for 2 h. Protein bands were detected by enhanced chemiluminescence.
10
Quantifying Small Intestinal Proteins
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Small intestinal mucosa were flushed with saline and then scratched. Total protein was extracted by RIPA lysis buffer (KeyGEN, China) with protease and phosphatase inhibitor cocktail (KeyGEN, China) and was quantified using bicinchoninic acid protein assay kit (Beyotime, China). Equivalent amount of small intestinal mucosal protein were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (Bio-Rad, United States) and separated by electrophoresis. Then, proteins were transferred onto 0.45 μm polyvinylidene fluoride membranes (Millipore, Ireland). After being blocked in 5 % skim milk powder (Beyotime, China) for 2 h, the membranes were incubated with primary antibodies to Smpd3 (Abcam, United Kingdom) and Sptlc2 (Invitrogen, United States) overnight, followed by incubation in horseradish peroxidase-conjugated secondary antibodies (Proteintech, China) for 60 min. The protein bands were visualized by ECL solution (Millipore, United States) and their density was assessed with LI-COR Odyssey Imager (LI-COR Biosciences, United States).
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