Western blot experiments were performed as previously described (22 (link)). The tissues and cells were lysed in RIPA buffer containing a protease inhibitor (PMSF) to harvest total proteins. The extraction of nuclear proteins was performed by using a commercially available Nuclear Extraction kit (#78833, Thermo Fisher Scientific, Logan, UT, United States) according to the manufacturer’s instructions. The proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrophoretically transferred to polyvinyl difluoride (PVDF) membranes. Following blockage of nonspecific binding sites with 5% nonfat dry milk for 1 h, the membranes were incubated with the appropriate primary antibodies against KPNA1, KPNA2, GAPDH, PCNA, Cyclin D1, IRF3 and Lamin B1 (1:1000 dilution) overnight at 4°C. After being rinsed with TBST, the membranes were incubated in a 1: 20,000 dilution of horseradish-peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Logan, UT, United States) for 1 h at room temperature. The immunoblotted proteins were visualized with enhanced chemiluminescence (ECL) reagents. Blots from at least three independent experiments were used for quantification purposes, and representative data are shown. The ImageJ software, an open-source image-processing program, was used to quantify the blots.
Nuclear extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nuclear Extraction Kit is a laboratory equipment designed to isolate and extract nuclear components, such as DNA and proteins, from cells. The kit provides a standardized and efficient method for separating the nuclear fraction from the cytoplasmic fraction, enabling researchers to study the specific contents and functions of the nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Emsa kit, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Pall biodyne b nylon membrane, by Pall Corporation (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti p65 antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Biosharp (1 mentions)
Haf008, by R&D Systems (1 mentions)
X omat film, by Kodak (1 mentions)
Hdac2, by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
Hdac5, by Abcam (1 mentions)
Histone h4, by Proteintech (1 mentions)
P iκbα, by Bioworld Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Histone h3, by Proteintech (1 mentions)
54 protocols using nuclear extraction kit
1
Comprehensive Western Blot Methodology
2
NF-κB Activation in Intestinal Cells
3
Protein Extraction and Western Blot Analysis
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Total proteins from cells were extracted with RIPA lysis buffer (Biosharp, China). Nuclear and cytosolic proteins were separated by nuclear extraction kit (Thermofisher Scientific, USA) according to the manufacturer's instructions. Protein concentration was determined using BCA Protein Assay Kit (Thermofisher Scientific, USA). WB was performed in a previous study [30 (link)]. Briefly, proteins were subjected from SDS-PAGE and transferred into nitrocellulose transfer membrane to incubate with 5% slim milk in PBS/0.05% Tween for 1 hr. The primary antibodies were added and incubated overnight at 4°C, followed by incubation with secondary antibodies (Jackson ImmunoResearch, UK) for 1 hr at room temperature. Proteins were imaged using an enhanced chemiluminescence (Perkin Elmer).
4
Western Blot Protein Analysis Protocol
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Whole cell lysates or nuclear proteins extracted using a nuclear extraction kit (Thermo Fisher Scientific Inc.) were prepared as detailed previously [30 (link)]. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The following primary antibodies were used for immunoblot analysis: α-SMA (ab5694), FOXM1 (ab180710), SOX9 (ab5535) from Abcam (Cambridge, MA, USA); gankyrin (12985S), Lamin B1 (13435S), phospho-AKT (9271S), PTEN (9188S) from Cell Signaling Technology (Danvers, MA); AKT (07-416) from Millipore (Burlington, MA); β-Actin (sc-1615), CK-7 (sc-70936), CK-19 (sc-33119), HNF4α (sc-6556), YAP (sc-101199) from Santa Cruz Biotechnology (Santa Cruz, CA). Membranes were blotted with primary antibody overnight at 4°C at a dilution of 1:1000 for all primary antibodies. Horseradish peroxidase–conjugated secondary antibodies for rabbit (HAF008; 1:3000) and goat (SC-2020; 1:3000) were obtained from R&D Systems (Minneapolis, MN) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Proteins were visualized with enhanced chemiluminescence reagents (ECL/ECL Plus, Amersham GE) and Kodak X-OMAT film.
5
Molecular Analysis of Prefrontal Cortex
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Samples from mice ACC were collected. Total proteins were lysed by M-PER Protein Extraction Buffer, and nuclear proteins of ACC were extracted with nuclear extraction kit (Thermo) according to the manufacturer’s instructions. Protein concentrations were determined using a BCA Kit. Equal amounts of protein aliquots were used for Western blot analysis to check the expression levels of LXRα, LXRβ, p50, p65, HDAC2, HDAC5, Iba-1 (Abcam), phosphorylated IκBα (p-IκBα, Bioworld Technology), phosphorylated extracellular regulated protein kinases (p-ERKs), ERK, phosphorylated p38 (p-p38), p38, phosphorylated c-Jun N-terminal kinase (p-JNK), JNK, GFAP (Cell Signaling Technology), AcH3 and AcH4 (Millipore), Histone H3, Histone H4, β-tubulin III (Proteintech), and β-actin (Sigma-Aldrich) served as a loading control.
6
Sp1 Binding Sites in TMBIM6 Promoter Analysis
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HT1080 and MCF-7 (source) cells were cultured in 100 mm plates in DMEM containing 10% FBS and grown for 24 h. Nuclear extracts were prepared using a Nuclear Extraction Kit (Thermo, Rockford, IL, USA). The oligonucleotides corresponding to the two Sp1 sites in the TMBIM6 core promoter region were synthesized and 3′-end biotin labeling was done using a Pierce™ Biotin 3′ End DNA Labeling Kit (Thermo, Rockford, IL, USA) and annealed. The sequence of the biotin-labeled probe corresponding to the Sp1 binding regions is listed in Table 2 . Then a LightShift Chemiluminescent EMSA kit (Thermo) was used according to the manufacturer’s protocol. In brief, 20 µL of 1× binding buffer, 50 ng/µL of Poly (dI.dC), 0.05% NP-40, 2 μg of nuclear extracts, 4 pmol of unlabeled competitor oligonucleotides, mutated unlabeled competitor oligonucleotides, and 20 fmol of labeled probe were applied and incubated at room temperature for 20 min. In the supershift assay, 0.2 μg of Sp1 (CST,#9389) antibody was added to the respective reactions and incubated for an additional 20 min at room temperature, normal rabbit IgG was used as a control. Then DNA-protein complexes were loaded onto a 6% non-denaturing polyacrylamide gel for blotting.
7
Subcellular Fractionation and Western Blotting
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Cells were lysed in SDS lysis buffer (100 mM NaCl, 500 mM Tris, pH 8.0, 10% SDS). For detection of NF-κB p100/52, p65 and c-Rel, a nuclear extraction kit (Thermo Scientific, MA, USA) was used to separate nuclear and cytoplasmic fractions. Cell extracts were subjected to 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies for Western blotting include: Galectin-3 (cat. 125402) (Biolegend, San Diego, USA), Galectin-1 (cat. Ab25138) and TSG101 (cat. ab30871; detects murine and human proteins) (Abcam, Cambridge, MA, USA), CD63, Erk1/2, NF-κB p65 (sc-8008 Figure 4G ) (Santa Cruz Biotechnology, USA), phospho-Erk1/2, NF-κB p65 (cat. 8242) and calreticulin (cat. 2891S; detects murine and human proteins) (Cell Signaling Technology, USA), NF-κB p100/52 (cat. 06–413) (Millipore, USA). Gapdh (Chemicon International, USA or Millipore MAB374), α-tubulin (Oncogene Science, Cambridge, USA), lamin A, Histone H3 (Cell Signaling Technology, USA) or β-actin (cat. GTX109639, GeneTex) were used as a loading control.
8
Protein Isolation and Western Blot Analysis
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After isolating the total protein from HeLa cells with RIPA buffer (10×, Cell Signaling, MA, USA), protein quantification was performed using a BCA assay kit (Sigma–Aldrich). Meanwhile, the nuclear extraction kit (Thermo Scientific, MA, USA) was applied to purify the nuclear proteins. The protein was separated via sodium dodecyl-sulfate polyacrylamide gel electrophoresis gel and subsequently transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk, membranes were incubated with primary antibodies specific for SOX2 (Invitrogen, MA, USA; #14-9811-82; 1 µg/mL), ALDH1 (Abcam, MA, USA; #ab9883; 0.5 µg/mL), Nanog (Invitrogen; #14-5,761-80; 2 µg/mL), p16 (Cell Signaling; #80772; 1:1,000), AE1 (Abcam; #ab9286; 1 µg/mL), Lamin A (Abcam; #ab108595; 1:12,000), and GAPDH (Beyotime, Jiangsu, China; #AF5009; 1:1,000) overnight. Next, membranes were washed three times before incubation with an horseradish peroxidase-conjugated secondary antibody (Beyotime, #A0208; 1:1,000) for 2 h. Finally, an enhanced chemiluminescence detection reagent (Sigma–Aldrich) was used to visualize protein bands. Densitometric analysis with ImageJ software was performed, and Lamin A and GAPDH were used as loading control for protein from nuclear and whole cell, respectively.
9
RNA Expression and Protein Analysis of Th Cell and DC Markers
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qPCR was performed as previously described10 (link). Total RNA was extracted from cells by using an RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. The mRNA levels of Dectin1, Il9, Ifng, Il4, Il5, Il13, Il17, Tnfsf15, Ox40l, Spi1, Irf4, Tbx21, Gata3 and Rorc by Th cells or DCs were analysed. Expression was normalized to the expression of the house-keeping gene Gapdh. Primer sets used for these analyses are listed in Supplementary Table 1 .
Western blot assay was performed as previously described10 (link). Anti-mouse phosphorylated (p)-Syk, Syk, pRaf1, Raf1, p-IKKα/β, IκB-α, p65, p50, c-Rel, RelB, p52, β-actin and HDAC1 antibodies were purchased from Cell Signaling Technology (CST). RIPA Buffer (cat #: 9806) were purchased from CST. Nuclear extraction kit (cat #: 78833) was purchased from Thermo Scientific. Images have been cropped for presentation. Full-size images are presented inSupplementary Figs 14 and 15 .
Western blot assay was performed as previously described10 (link). Anti-mouse phosphorylated (p)-Syk, Syk, pRaf1, Raf1, p-IKKα/β, IκB-α, p65, p50, c-Rel, RelB, p52, β-actin and HDAC1 antibodies were purchased from Cell Signaling Technology (CST). RIPA Buffer (cat #: 9806) were purchased from CST. Nuclear extraction kit (cat #: 78833) was purchased from Thermo Scientific. Images have been cropped for presentation. Full-size images are presented in
10
Quantitative Western Blot Analysis
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Western blotting was performed as described previously.24 Briefly, total protein was extracted from PDAC cell lines in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Rockford, IL, USA) and nuclear proteins were extracted with the Nuclear Extraction Kit according to the manufacturers’ protocol. Aliquots of total protein (12 μg) were electrophoresed on sodium dodecyl sulfate polyacrylamide gels, 10% Tris‐HCL gels (Bio‐Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio‐Rad Laboratories) and incubated with primary antibodies for 1 hour. Proteins were detected with anti‐HDAC1 antibody (1:200 dilution; Santa Cruz Biotechnology), anti‐SNAIL antibody (1:2000 dilution; Abcam), anti‐ZEB1 antibody (1:1000 dilution; Santa Cruz Biotechnology), anti‐Cytokeratin 19 antibody (1:200 dilution; Santa Cruz Biotechnology), anti‐Histone H3 (1:2000; Cell Signaling Technology, Danvers, MA, USA) and anti–β‐actin antibody (diluted 1:4000; Sigma, Tokyo, Japan). The expression relative to actin expression was depicted as a column and measured with ImageJ software (rsb.info.nih.gov/ij).
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