MDBK cells were pretreated by LiCl at a final concentration of 20 mM for 24 h, and then were infected by 1 MOI BPIV3 for 24 h. After that, cells were harvested and the nucleus and cytoplasm were separated according to the manufacturer’s protocol of Minute™ Cytoplasmic and Nuclear Fractionation kit (Invent Biotechnologies, Inc) and then subjected to western blot analysis. β-actin and Lamin B1 were used as internal references of cytoplasmic and nucleus protein, respectively.
Minute cytoplasmic and nuclear fractionation kit
Manufactured by Invent Biotechnologies
Sourced in United States, China
The Minute™ Cytoplasmic and Nuclear Fractionation kit is a lab equipment product designed to separate and extract cytoplasmic and nuclear fractions from eukaryotic cells. The kit provides a simple and efficient method for the isolation of these cellular components.
Automatically generated - may contain errors
Lab products found in correlation
β mhc, by Abcam (1 mentions)
Lamin b1, by Proteintech (1 mentions)
Tubulin, by Beyotime (1 mentions)
Odyssey infrared fluorescence imaging system, by LI COR (1 mentions)
G3bp2, by Affinity Biosciences (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Gapdh, by Beyotime (1 mentions)
Super bradford protein assay kit, by CWBIO (1 mentions)
β actin, by Merck Group (1 mentions)
Ab16502, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
6 protocols using minute cytoplasmic and nuclear fractionation kit
1
BPIV3 Infection in LiCl-Pretreated MDBK Cells
2
Evaluating Nrf2 and SIRT1 Signaling
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Nuclear extracts were prepared using the Minute Cytoplasmic and Nuclear Fractionation Kit (Invent Biotechnologies Inc., Plymouth, MN). The nuclear fractionations were then subjected to Western blotting analysis using anti-Nrf2 antibody. Total proteins were subjected to Western blotting analysis using anti-SIRT1, anti-eNOS,anti-p-eNOS, and anti-HO-1 antibody. Western blot analysis was performed as previously described (Jiang et al., 2019 (link)).
3
Subcellular Fractionation and NF-κB Analysis
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The nuclear and cytoplasmic fractions were separately extracted using the Minute™ Cytoplasmic and Nuclear Fractionation Kit (SC-003, Invent Biotechnologies, USA). β-Actin was used as the cytoplasmic control, and Lamin B1 was used as the nuclear control for western blotting analysis. Nuclear p65 DNA-binding activity was detected using NF-κB p65 transcription factor assays (ab210613, Abcam, UK).
4
Protein Extraction and Western Blot Analysis
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Protein extraction and western blotting were performed as previously reported.56 (link) Briefly, total proteins were extracted from lysis buffer containing protease inhibitor cocktail (Kangchen, cat. no. KC-415), and nuclear or cytoplasmic proteins were extracted using the Minute Cytoplasmic and Nuclear Fractionation kit (Invent, cat. no. SC-003). Proteins (30 μg) were subjected to SDS-PAGE using polyacrylamide gels. Immunoblotting was performed using specific antibodies (ANP, Beyotime, cat. no. AF7608; BNP, Abcam, cat. no. ab236101; β-MHC, Abcam, cat. no. ab172967; NF-κB p65, Cell Signaling, cat. no. D14E12; IκBα, Cell Signaling, cat. no. 9242; G3BP2, Affinity Biosciences, cat. no. DF4387, TUBULIN, Beyotime, cat. no.AF1216; GAPDH, Beyotime, cat. no. AF1186 and Lamin B1, Proteintech, cat. no.12987-1-AP) to evaluate the expression of proteins. The image was acquired using the Odyssey infrared fluorescence imaging system (Li-Cor, Lincoln, NE). Band intensity was quantified by a densitometric analysis using ImageJ software (National Institutes of Health).
5
Investigating Nuclear Translocation of NF-κB in BP-Exposed Macrophages
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Macrophage-like dTHP-1 cells were treated with BP nanomaterials (100 μg ml−1) and BP–corona complexes for 6 h. Then the cells were washed with PBS three times and collected. The cytoplasm and nuclear extracts were prepared using the Minute™ Cytoplasmic and Nuclear Fractionation Kit (Invent Biotechnologies, Inc), and the protein concentration was determined by the Super-Bradford Protein Assay Kit (CWBiotech, Inc, Beijing, China). The expression levels of lκB in cytoplasm extracts and p65 in nuclear extracts were determined by WB according to previous literature54 (link). Briefly, the extracts were first separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad, CA, USA). The membrane was blocked with 5% BSA in PBS at 25 °C for 2 h and then incubated with antibodies at 4 °C overnight. The expression of β-actin was used as the internal standard. The uncropped WB images were included in Supplementary Figure 12 . The sources of primary antibody against the following protein were: β-actin (A1978, 1:10,000 for WB) from sigma; p65 in nuclear (ab16502, 1:1000 for WB), and lκB (15095, 1:1000 for WB) from Abcam. The appropriate secondary antibodies (1:10,000 for WB) were purchased from Abcam.
6
Subcellular Fractionation and Western Blot Analysis
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Subcellular fractions of nuclei or cytoplasm were isolated by Minute™ Cytoplasmic and Nuclear Fractionation kit (Invent Biotechnologies, Inc.; cat. no. #SC-003) according to the manufacturers' protocol. The extracted proteins were examined via western blot using β-actin (Cell Signaling Technology, Inc. cat. no. #4970S) serving as marker for total and cytoplasmic protein, Lamin A antibody (CSignaling Technology, Inc.; cat. no. #86846S) serving as marker for nuclear protein. Western blot was performed as described in a previous study (21 (link)). Primary antibody against p53 (cat. no. #60283-2-Ig, 1:1,000), Kras (cat. no. #12063-1-AP, 1:1,000), pan-Ras (cat. no. #60309-1-Ig, 1:1,000), AKT (cat. no. #60203-2-Ig, 1:1,000), phosphorylated (p-)AKT (Ser473; cat. no. #80455-1-RR, 1:1,000), p-AKT (Thr308; cat. no. #29163-1-AP, 1:1,000), c-MYC (cat. no. #67447-1-Ig; 1:1,000), LDHA (cat. no. #21799-1-AP, 1:1,000) and GAPDH (cat. no. #60004-1-Ig, 1:2,000) were obtained from ProteinTech Group, Inc.; p-c-MYC (Ser293; cat. no. #PA5-105447, 1:2,000) was obtained from Invitrogen (Thermo Fisher Scientific, Inc.) and p-MYC (T58 + S62; cat. no. #13342, 1:1,000) was obtained from Signalway Antibody LLC. Antibodies against E-cadherin, N-cadherin, vimentin, Snail and Slug were obtained from EMT Antibody Sampler kit (cat. no. #9782; Cell Signaling Technology, Inc.; 1:1,000).
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