SDS‐PAGE was conducted as previously reported with modifications.[78 ] Brain samples were homogenized in RIPA buffer and protease inhibitor cocktail. Cytoplasmic and nuclear fractions were prepared from primary astrocytes which were treated with different drug and different groups of mice brain using Minute Cytoplasmic & Nuclear Extraction Kits (SC‐003, Invent Biotechnologies, USA). The primary antibodies used included AQP4 (rabbit, 1:1000, Abcam, ab46182), AQP4 (rabbit, 1:1000, Alomone, AQP‐004), and anti‐β‐actin (mouse, 1:2000, Cell Signaling Technology, 3700s), GFAP (rabbit, 1:1000, Millipore, Mab360), LC3B(rabbit, 1:1000, Cell Signaling Technology, 3868s), p62 (rabbit, 1:1000, Sigma, p0067), p‐mTOR (mouse, 1:1000, Cell Signaling Technology, 5536s), mTOR (mouse, 1:1000, Cell Signaling Technology, 2983s), p‐ULK1 (mouse, 1:1000, Cell Signaling Technology, 14202), ULK1 (mouse, 1:1000, Cell Signaling Technology, 8054), LAMP1 (mouse, 1:1000, Invitrogen,14‐1071‐82), PPAR‐γ (rabbit, 1:1000, Cell Signaling Technology, 2443s), GAPDH (mouse, 1:2000, Abcam, ab9484), LaminB1 (mouse, 1:1000, Abcam, ab220797), IgG (rabbit, 1:1000, Proteintech, 2729p), Nav1.6 (mouse, 1:1000, Abcam, ab65166), Nav1.6 (rabbit, 1:200, Sigma, WH0006334M4), pERK1/2 (rabbit, 1:1000, Cell Signaling Technology, 3179S), pJNK (mouse, 1:2000, Cell Signaling Technology, 9255S), p‐P38 (mouse, 1:2000, Cell Signaling Technology, 9216S), pAKT (rabbit, 1:2000, Cell Signaling Technology, 4060S), pGSK3β (rabbit, 1:1000, Cell Signaling Technology, 9323S) and the sample‐loaded membranes were incubated overnight at 4 degree and then post 8–12 h of incubation treated with secondary antibody, goat anti‐rabbit (1:5000, Thermo, A16104) and goat anti‐mouse IgG (1:5000, Thermo, 31430), along with electrochemiluminescence (ECL, Millipore) reagent. BIO‐RAD (Hercules, CA, USA) gel analysis software was used to detect band signals.
Minute cytoplasmic nuclear extraction kit
Manufactured by Invent Biotechnologies
Sourced in United States
About the product
The Minute™ Cytoplasmic & Nuclear Extraction Kits are laboratory equipment designed to efficiently isolate and extract cytoplasmic and nuclear fractions from various cell types. The kits provide a simple and reliable method for the separation of these cellular components.
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11 protocols using «minute cytoplasmic nuclear extraction kit»
1
Protein Expression Analysis in Astrocytes
2
Cytoplasmic and Nuclear Protein Extraction
Cytoplasmic and nuclear proteins were separated using Minute Cytoplasmic & Nuclear Extraction Kits (SC-003, Invent, USA) according to the manufacturer’s protocol. Briefly, cytoplasmic extraction buffer was added to the cell plates, and the plates were incubated on ice for 5 min, followed by a vigorous vortex for 15 s and centrifugation for 5 min at top speed. The supernatant (cytoplasmic fraction) was transferred to a new tube, and nuclear extraction buffer was added to the pellets. The pellets were vortexed vigorously for 15 s and then incubated on ice for 1 min, and this process was repeated for four times. The nuclear extract was transferred to a filter cartridge with a collection tube and centrifuged at top speed for 30 s. The cytoplasmic and nuclear proteins were further subjected to Western blot analysis.
3
Cell Fractionation and Western Blot
Cytoplasmic and nuclear fractions were prepared from 5 × 107 cells by using Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent Biotechnologies, USA). Briefly, cells were harvested and lysed with cytoplasmic/nuclear lysis buffer in order. After centrifugation, the supernatants were collected as the cytoplasmic/nuclear fraction and then analyzed using western blot.
4
Quantifying HDAC Activity in Cells
HSV-2–infected or romidepsin-treated cells were fractionated with Minute Cytoplasmic & Nuclear Extraction Kits (SC-003, Invent Biotechnologies Inc.), and at least 0.5 μg of nuclear extract was used to determine HDAC activity (Epigenase HDAC Activity/Inhibition Direct Assay Kit, P-4034-96, EpigenTek). Absorbance was measured using a SpectraMax M5e microplate reader and SoftMax Pro 7.1 GxP software (Molecular Devices).
5
Protein Extraction and Western Blot Analysis
The total protein was extracted from cells lysed with sodium dodecyl sulfate (SDS, Beyotime Biotechnology, Shanghai, China) lysate containing 1% phenylmethanesulfonyl fluoride (Beyotime Biotechnology) and 1% phosphatase inhibitor. The cytoplasmic and nuclear proteins were isolated with the Minute™ Cytoplasmic & Nuclear Extraction Kit (#SC-003; Invent Biotechnologies, Inc, Minnesota, USA) according to the manufacturer's instructions. The protein concentration was measured with the BCA protein assay kit (Beyotime Biotechnology). Protein samples were run on SDS-polyacrylamide gel electrophoresis (SDS- PAGE) and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was incubated with the primary antibody at 4 ℃ overnight. The following antibodies were used in the study: anti-CDK14 antibody (1:1000 dilution, Santa Cruz Biotechnology, Inc, Dallas, Texas USA), anti-MDR1 (1:5000 dilution, P-gp, Cell Signaling Technology, Inc., Boston, MA, USA), and anti-β-actin (1:5000 dilution, Proteintech, Wuhan, China). The secondary antibodies of the anti-mouse IgG or anti-rabbit IgG were used at RT for 1 h. The protein bands were photographed by the chemiluminescence imaging system (Tanon Science & Technology, Shanghai, China).
Top 5 protocols citing «minute cytoplasmic nuclear extraction kit»
1
Protein Extraction and Quantification for Western Blot Analysis
Fresh heart tissue was prepared and total protein was extracted by the Protein extraction kit (KGP2100, KeyGEN BioTECH, China). Cytoplasmic and nuclear protein were separated by Minute™ Cytoplasmic & Nuclear Extraction Kits (SC-003, Invent, USA) according to the manufacturer's instructions. Protein concentration was then quantitated with the Bradford protein quantitation assay (KGA803, KeyGEN BioTECH, China). 30 μg of total protein per lane was separated via SDS-PAGE gel and immunoblotted with primary antibodies against histone H3 (#4499, Cell Signaling Technology, USA), α-Tubulin (#2125, Cell Signaling Technology, USA), TnI (recognize both with mouse ssTnI and cTnI) (approved by Florida Atlantic University, as described in our previous studies12 (link),13 (link),23 (link)), GAPDH (Sino Biological, China), SERCA2a (#9580, Cell Signaling Technology, USA). Secondary antibodies were anti-rabbit (Sino Biological, China) or anti-mouse IgG HRP (Sino Biological, China), which were linked to the whole antibody. The immune-reactive protein bands were visualized and band intensity was analyzed and quantified by using Amersham Imager 600 system (GE Healthcare, USA).
2
Protein Extraction and Fractionation from Endometrial Samples
Total protein extraction from endometrial tissue samples and EC cells was prepared as previously reported [35 (link)]. Nuclear and cytosolic fractions were isolated using Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent Biotechnologies, Inc., Plymouth, MN, USA). Primary antibodies were used at the following dilutions: monoclonal anti-GAPDH-horseradish peroxidase conjugates at 1 : 10,000 (GNI, Japan, Cat#GNI4310-GH-S), anti-EIF1AX antibodies at 1 : 1,000, anti-Snail antibodies at 1 : 1,000 (Proteintech, USA, Cat#26183-1-AP), anti-E-cadherin antibodies at 1 : 1,000 (Cell Signaling Technologies, USA, Cat#14472), anti-IPO13 antibodies at 1 : 1,000 (Novus Biologicals, Cat#NBP1-31508), and anti-XPO1 antibodies at 1 : 1,000 (Santa Cruz Biotechnology, Cat#sc-74454).
3
Cytoplasmic and Nuclear Fractionation
Cytoplasmic and nuclear fractions were prepared from 5 × 107 cells by using Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent Biotechnologies, USA). Briefly, cells were washed with ice-cold PBS and lysed for 30 min on ice with cytoplasmic lysis buffer. The lysates were centrifuged at 14000 rpm for 5 min at 4 °C, and the supernatants (cytoplasmic fractionation) were collected in a fresh tube kept on ice. The pellet was resuspended with nuclear lysis buffer. The homogenate was incubated on ice for 30 min on ice and centrifuged at 14000 rpm for 5 min at 4 °C. Supernatant was collected as the nuclear fraction. The cytoplasmic and nuclear fractions were then analyzed using western blot.
4
Subcellular Fractionation and Western Blot Analysis
Cytoplasmic and nuclear fractions were prepared from cells using Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent Biotechnologies, USA) according to the manufacturer's instructions.
Whole cell lysates were prepared in RIPA buffer (Thermo Fisher Scientific) containing protease/phosphatase inhibitor cocktail (Cell Signalling). Protein samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The membrane was incubated with the following primary antibodies (all from Abcam): anti-SMYD3 (ab187149, 1:1000), anti-H3K4me3 (ab8580, 1:1000), anti-ANKHD1 (ab117788, 1:2000), anti-CDK2 (ab32147, 1:1000), anti-H3 (ab1791, 1:1000), and anti-GAPDH (ab181602, 1:5000). After washing the membrane with TBST three times, it was incubated with HRP-labelled goat anti-mouse/rabbit IgG (1:5000, Abcam) for 2 h at room temperature. Specific signals were detected using the enhanced chemiluminescence (ECL, Millipore, MA, USA) method and analysed with ImageJ software.
Whole cell lysates were prepared in RIPA buffer (Thermo Fisher Scientific) containing protease/phosphatase inhibitor cocktail (Cell Signalling). Protein samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The membrane was incubated with the following primary antibodies (all from Abcam): anti-SMYD3 (ab187149, 1:1000), anti-H3K4me3 (ab8580, 1:1000), anti-ANKHD1 (ab117788, 1:2000), anti-CDK2 (ab32147, 1:1000), anti-H3 (ab1791, 1:1000), and anti-GAPDH (ab181602, 1:5000). After washing the membrane with TBST three times, it was incubated with HRP-labelled goat anti-mouse/rabbit IgG (1:5000, Abcam) for 2 h at room temperature. Specific signals were detected using the enhanced chemiluminescence (ECL, Millipore, MA, USA) method and analysed with ImageJ software.
5
Protein Extraction and Western Blot Analysis
Whole-cell and nuclear protein (Minute™ Cytoplasmic & Nuclear Extraction Kits; Invent Biotechnologies, Eden Prairie, MN, USA) was separated using 7.5% or 10.0% SDS-PAGE, transferred onto nitrocellulose membranes, and probed with antibodies against Nrf2 (1:15,000; Abcam), p-Nrf2 (Ser40) (1:15,000; Abcam), Keap1 (1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated p62 (phospho-p62, Ser351, 1:1000; Medical & Biological Laboratories Co., LTD, Woburn, MA, USA), STAT3 (1:1000; Cell Signaling Technology), phosphorylated STAT3 (phospho-STAT3, Thy705, 1:500; Cell Signaling Technology), Lamin B1 (1:10,000; Abcam), and β-actin (1:10,000; Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation at 4 °C, the membranes were washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies and visualized using an ECL prime detection kit (GE Healthcare, Buckinghamshire, UK).
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