Total protein was obtained using RIPA lysis buffer. Cell membrane, cytoplasm and mitochondrial proteins were obtained using the cell membrane protein and cytoplasmic protein extraction kit (Beyotime) and the cell mitochondrial isolation kit (Beyotime). BCA protein assay Kit (APPLYGEN) was used to quantitate the protein levels. The primary antibody information used is as follows: anti-CLDN10 (1:1000; Abcam, ab52234), anti-Flag (1:1000; CST, 14793S), anti-ATP5O (1:2000; Abcam, ab110276), anti-Acetyl-ATP5O (1:200; Abcam, ab214339), anti-SIRT3 (1:1000; Abcam, ab217319), anti-NDUFS2 (1:5000; Abcam, ab192022), anti-Cleaved-Caspase 3 (1:1000; Affinity, AF7022), anti-E-cadherin (1:10000; Abcam, ab40772), anti-N-cadherin (1:5000, Abcam, ab76011), anti-SDHB (1:5000, Proteintech, China), anti-GAPDH (1:8000, Proteintech, China), anti-ATP1A1 (1:8000, Proteintech, China) and anti-COX Ⅳ (1:8000, Proteintech, China).
Cell membrane protein and cytoplasmic protein extraction kit
Manufactured by Beyotime
Sourced in China
The Cell membrane protein and cytoplasmic protein extraction kit is a laboratory tool designed to extract and separate cellular proteins from the cell membrane and cytoplasm. The kit provides a standardized protocol and reagents to facilitate the isolation and purification of these distinct protein fractions from cell samples.
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Lab products found in correlation
Anti e cadherin, by Abcam (1 mentions)
Anti sirt3, by Abcam (1 mentions)
Anti atp1a1, by Proteintech (1 mentions)
Anti sdhb, by Proteintech (1 mentions)
Anti cleaved caspase 3, by Affinity Biosciences (1 mentions)
Cell mitochondrial isolation kit, by Beyotime (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Bca protein assay kit, by Applygen (1 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Beyotime (1 mentions)
5 protocols using cell membrane protein and cytoplasmic protein extraction kit
1
Subcellular Protein Extraction and Quantification
2
Membrane Protein Fractionation Protocol
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Membrane protein fractions were separated using a Cell membrane protein and cytoplasmic protein extraction kit (Beyotime) according to the manufacturer’s instructions. Briefly, HCC cells were collected by cell scrapers, followed by three washes with cold PBS. Then, 1 mL of membrane protein extraction reagent A with PMSF was added. Cells were placed in an ice bath for 10 to 15 min. Then, cell lysates were centrifuged (700 × g) at 4 °C for 10 min, followed centrifugation (14,000 × g) for 30 min at 4 °C. The supernatant (cytoplasmic protein) was discarded to obtain membrane fraction-enriched lysates. Afterwards, 200 μL of membrane protein extraction reagent B was added, and cell lysates were resuspended and forcefully vortexed at high speed for 5 s, followed by incubation in an ice bath for 5 to 10 min. The vortexing and ice bath incubation steps were then repeated twice. The lysates were centrifuged (14,000 × g) at 4 °C for 5 min. The membrane proteins were enriched in the supernatants.
3
Cell Membrane and Cytoplasmic Protein Extraction
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The treated cells were collected, and then cell membrane and cytoplasmic proteins were extracted by a cell membrane protein and cytoplasmic protein extraction kit (Beyotime) in accordance with the manufacturer’s instructions.
4
Isolation and Extraction of Cell Membrane Proteins
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The digested TE10 cells (1 × 108) were collected and centrifuged at 700 g for 5 min. The cells were suspended in pre-cooled phosphate buffer saline (PBS) buffer (pH = 7.4) and then centrifuged at 700 g for 5 min. According to the instruction of Cell membrane protein and cytoplasmic protein extraction kit (Beyotime, China), specifically, TE10 cells were collected and washed 3 times with pre-cooled PBS, and then resuspended with 1 mL reagent A. After ice bathing for 15 min, the cell suspension was placed in liquid nitrogen and room temperature (RT) successively, and then freeze-thaw for twice. After that, it was centrifuged at 700 g, 4°C for 10 min, then the supernatant was centrifuged 14,000 g, 4°C for 30 min. Finally, the collected cell pellets were suspended with membrane protein extraction reagent containing Phenylmethylsulfonyl fluoride (PMSF). The cells were ice-bathed in this hypotonic lysis buffer for 10–15 min and then lysed with ultrasound. Cell lysate was centrifuged at 700 g for 10 min and the supernatant was then centrifuged at 14,000 g for 30 min. The precipitate was the extracted cell membrane.
5
Multifunctional PLGA Nanoparticles for Cancer Therapy
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Carboxy-terminated polylactic acid/glycolic acid PLGA (PLGA-COOH polymerization ratio: 50:50, molecular weight: 12,000 Da) was purchased from Jinan Daigang Biotechnology Co., Ltd. (Jinan, China). Hematoporphyrin monomethyl ether (HMME) was purchased from Shanghai DB Chemical Technology Co., Ltd. (Shanghai, China). Oleic-acid-modified superparamagnetic iron oxide (SPIO) nanoparticles (d = 10 nm) were purchased from Ocean Nano Tech, Inc., (AR, USA). The cell membrane protein and cytoplasmic protein extraction kit and phenylmethanesulfonyl fluoride (PMSF) were purchased from Beyotime (Shanghai, China). Calcein-AM, PI and CCK-8 assay kits were purchased from Dojindo Laboratories (Kumamoto, Japan). Anti-mouse PD-1 (CD279, Lot: 78012ON, Catalog No. BE0146) was obtained from Bioxcell (USA). ELISA kits were purchased from Elabscience Biotechnology Co., Ltd (Wuhan China). Antibodies to cell surface markers for flow cytometry analysis were purchased from BioLegend, Inc., (CA, USA). All unspecified reagents used were of analytical grade or better.
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