Cellular fractions were isolated using the Nuclear and Cytoplasmic Protein Extraction Kit (20126ES50; Yeasen, Shanghai, China). The cells were washed with 1× PBS and lysed in RIPA lysis buffer containing protease inhibitors, and the protein levels in the lysates were quantified using an enhanced bicinchoninic acid assay (BCA) kit (P0012, Beyotime, Shanghai, China). Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and immunoblotted with the indicated antibodies. For immunoprecipitation assays, protein lysates were incubated with anti-ARID1A, anti-YAP, anti-BRG, anti-TEAD, anti-Flag, or normal IgG antibody at 4 °C overnight with rotation. On day 2, the mixture was incubated with protein A/G beads at room temperature for 2–3 h. After washing three times with lysis buffer, the beads were boiled and subjected to Western blotting. The antibodies used were as follows: ARID1A (ab272905, Abcam, Cambridge, UK); YAP (#14074, CST, Fall River, MA, USA); p-YAP (#13008, CST); BRG1 (ab110641, Abcam); TEAD (ab133533, Abcam); E-cadherin (#14472, CST); N-cadherin (#13116, CST); MMP2 (ab92536, Abcam); MMP-9 (ab283575, Abcam); Snail (#3879, CST); Vimentin (ab92547, Abcam); GAPDH (60004-1-Ig, Proteintech, Chicago, IL, USA); Histone H3 (ab17917, Abcam), Flag (20543-1-AP, Proteintech).
Nuclear and cytoplasmic protein extraction kit
Manufactured by Yeasen
Sourced in China
The Nuclear and Cytoplasmic Protein Extraction Kit is a laboratory tool designed to separate and extract nuclear and cytoplasmic proteins from cells. It provides a standardized and efficient method for obtaining these cellular fractions for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab110641, by Abcam (1 mentions)
Ab283575, by Abcam (1 mentions)
Ab133533, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Skim milk, by Beyotime (1 mentions)
Page gel quick preparation kit, by Yeasen (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Bca protein quantification kit, by Yeasen (1 mentions)
Ab134175, by Abcam (1 mentions)
C myc ab32072, by Abcam (1 mentions)
Proliferating cell nuclear antigen (pcna), by ABclonal (1 mentions)
Lamina c, by ABclonal (1 mentions)
β catenin 51067 2 ap, by Proteintech (1 mentions)
Ripa lysis buffer, by Yeasen (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Potent ecl kit, by Yeasen (1 mentions)
8 protocols using nuclear and cytoplasmic protein extraction kit
1
Cellular Fractionation and Protein Analysis
2
Nuclear and Cytoplasmic Protein Extraction
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A total of 2 × 106 T24 cells were lysed using Nuclear and Cytoplasmic Protein Extraction Kit (Cat# 20126ES50;Yeasen). Samples were normalized for protein concentration using Pierce BCA Protein Assay. Then, 10 μg of each cytosolic and nuclear extract sample were analyzed by SDS-PAGE and Western blotting using specific antibodies.
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using RIPA lysis buffer. The nuclear and cytoplasmic protein was extracted using a commercial kit (Nuclear and Cytoplasmic Protein Extraction Kit, Yeasen Biotechnology, Shanghai, China). The protein concentration was determined with a BCA kit, and 30 μg of protein was loaded and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with the primary antibodies at 4 °C overnight. The next day, after three washings in PBST (1% Tween diluted in PBS), the membranes were incubated with secondary antibodies at room temperature for 1 h. The membranes were finally scanned with an Odyssey (Licor, Lincoln, NE, USA).
4
Protein Analysis by Western Blotting
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Proteins were extracted with Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, China), and their concentrations were checked with BCA Protein Quantification Kit (Yeasen, China). After being separated using SDS-PAGE with PAGE Gel Quick Preparation Kit (12.5% and 10%) (Yeasen, China) and transferred onto the PVDF membrane (GE, USA), the unreacted sites were blocked with 5% skim milk (Beyotime, China) for 2 h and the primary antibodies against SLC7A5 (1 : 200, goat, ab99419), β-actin (1 : 500, goat, ab8229; 1 : 1000, rabbit, ab8227), p-mTOR (phospho-S2448, 1 : 1000, rabbit, ab109268), mTOR (1 : 1000, rabbit, ab32028), p-S6K1 (phospho-S424, 1 : 500, rabbit, ab131436), S6K1 (1 : 5000, rabbit, ab32529), p-4EBP (1 : 500, rabbit, ab47365), and 4EBP (1 : 2000, rabbit, ab32024) (Abcam, USA) were added and the samples were incubated at 4°C for 12 h. After being rinsed three times with TBST (Yeasen, China), the secondary HRP-conjugated donkey anti-goat antibody (1 : 1000, ab6885) or goat anti-rabbit antibody (1 : 2000, ab6721) (Abcam, USA) was administrated to the membrane and maintained for 1 h at room temperature. Next, the bands were washed with TBS-T and visualized using the 4CN HRP kit (Leagene, China). The relative expression levels were obtained via ImageJ 1.53f (NIH, USA).
5
Comprehensive Protein Extraction and Analysis
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Total protein was extracted using RIPA lysis buffer from Yeasen (China). Cytoplasmic and nuclear protein extraction was carried out using the Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, China). ACTIN and LaminA/C were used as internal controls for cytoplasmic and nuclear proteins, respectively. Primary antibodies used in this study were, c-Myc(ab32072, Abcam, USA), PCNA (A0264, ABclonal, China), SRSF1 (sc-33652, Santa Cruz Biotechnology, China), β-catenin (51067-2-AP, Proteintech, USA), cyclin D1(ab134175, Abcam, USA), LaminA/C (A0249, ABclonal, China), and ACTIN (AC026, ABclonal, China).
6
Subcellular Protein Fractionation Protocol
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Nuclear and cytoplasmic proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, Shanghai, China) according to the manufacturer’s instructions. In brief, cells were washed and harvested by scraping in cold PBS. Cell pellets were obtained after centrifugation. 200 µL of reagent A containing PMSF was added per 20 µL cell precipitation. The sample was vortexed vigorously (highest setting on vortex) for 5 s, then incubated for 15 min in an ice bath. Subsequently, 10 µL of cytosolic protein extraction reagent B was added to the samples and the mixture was vortexed vigorously for 5 s, then incubated for 1 min in an ice bath. Samples were centrifuged at 12,000–16,000g for 5 min at 4 °C. The supernatant was the extracted cytoplasmic protein. 50µL of reagent C containing PMSF was added to the precipitate, then the mixture was incubated for 30 min in an ice bath. Samples were centrifuged at 12,000–16,000g for 10 min at 4 °C. The supernatant was the extracted nuclear protein, which was immediately transferred into a pre-cooled tube.
7
Protein Expression Analysis in Cell Lysates
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The cells were lysed in RIPA buffer. Equal amounts of total protein were resolved, transferred to PVDF membranes (Millipore) and immunoblotted with the specified primary antibodies. Membranes were hybridized with the following primary antibodies: AhR (Abcam, #ab190797, 1:1000), NIS (Abcam, #ab242007, 1:1000), IGF2BP2 (Abcam, #ab124930, 1:2000), Histone 3 (Abcam, # ab267372, 1:1000) and β-actin (Abcam, #ab8227, 1:1000). Species-specific HRP-conjugated antibodies (1:5000; Cell Signaling Technology) were used to hybridize membranes. The cytoplasm and nuclear proteins were recovered and separated using the nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, Shanghai, China), following cell lysis. The Potent ECL kit (Yeasen, Shanghai, China) was used to visualize the bands.
8
Extracting Nucleus and Cytoplasmic Proteins from H7N9-Infected Cells
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Flag-MYH9-H1299 cells and NC-H1299 (4 × 106 cells per 10-cm dish) were infected with H7N9 (MOI = 5) for 1, 2, 3, and 6 h. Nucleus and cytoplasm were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen) according to the manufacturer's instructions. In brief, cells were rinsed twice with cold PBS and were then scraped off with a cell scraper. Cells were collected centrifugally, and the supernatant was aspirated as far as possible, leaving cell precipitation for later use. 200 μL phenylmethanesulfonyl fluoride (PMSF)-containing reagent A was added for every 20 μL cell precipitates, and the cell precipitates were completely suspended and bathed in ice for 10 min 10 μL of cytoplasmic protein extraction reagent B was added and then centrifuged. The supernatant was the extracted cytoplasmic protein. For the precipitates, the residual supernatant was completely sucked up and 50 μL PMSF-containing reagent C was added. Then the mixture was completely suspended and bathed in ice for 30 min. The supernatant was collected centrifugally, namely, the nuclear protein obtained by extraction. All samples were mixed with SDS loading buffer and boiled for western blotting.
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