Protein expression of the A1 astrocyte marker C3 (complement 3) and A2 astrocyte marker S100A10 was determined by western blot. Total protein was extracted from astrocytes using the whole cell lysis kit (Keygen, Nanjing, China), and protein concentration was determined using the BCA assay (Thermo Fisher Scientific, New York, USA). Equal amounts of proteins per sample were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Massachusetts, USA). The blots were then incubated with primary antibodies against C3 (1:50; Abcam, Cambridge, UK), S100A10 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000; Proteintech, Wuhan, China).
Whole cell lysis kit
Manufactured by Keygen Biotech
Sourced in China
The Whole Cell Lysis Kit is a laboratory tool designed to efficiently disrupt and lyse cells, releasing their cellular contents. The kit provides a standardized and effective method for preparing samples for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gapdh, by Proteintech (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Anti nf κb p65, by ABclonal (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Anti c ebpβ, by Abcam (1 mentions)
Anti il 1β, by ABclonal (1 mentions)
Bca protein quantitation kit, by Keygen Biotech (1 mentions)
Anti gapdh 6c5, by Abcam (1 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti osterix, by Abcam (1 mentions)
Anti cd63, by Abcam (1 mentions)
7 protocols using whole cell lysis kit
1
Astrocyte Marker Protein Expression
2
Western Blot Analysis of Autophagy Markers
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The PLCs were harvested for protein extraction using a whole cell lysis kit (Nanjing KeyGen Biotech, Nanjing, China). The total protein concentration of the samples was determined with a BCA protein assay kit (Nanjing KeyGen Biotech, Nanjing, China) according to the manufacturer’s instructions. Equal amounts of protein (20 μg) were separated with 10% SDS-PAGE gels and transferred to PVDF membranes by electroblotting. The transferred membranes were blocked with 10% nonfat milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: BCL-2 (1:1000), BAX (1:1000), ATG5 (1:1000), ATG16L (1:1000), ATG4B (1:1000), P62 antibody (1:500), LC3-II (1:1000), and β-actin (1:5000). Next, the membranes were incubated with HRP-linked goat anti-mouse or goat anti-rabbit IgG (1:5000 dilutions) for 1 h at room temperature. The signal was measured using an enhanced chemiluminescence (ECL) kit (New Cell and Molecular Biotech Co. Ltd., Suzhou, China), and the protein band intensity was quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).
3
Protein extraction and Western blot analysis
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The Whole Cell Lysis Kit and the BCA Protein Quantitation Kit (KeyGEN Bio TECH) were used to achieve cell protein extraction and quantification. Then Western blotting (WB) was performed according to a previously described standard method17 (link) using anti-C/EBPβ (1:1000, Cat#ab32358, Abcam), anti-iNOS (1:1000, Cat#OM641716, Omnimabs), anti-NOD2 (1:1000, Cat#A15992, ABclonal), anti-NOD1 (1:1000, Cat#DF6378, Affinity), anti-phospho-RIPK2 (1:500, Cat#AF0049, Affinity), anti-RIPK2 (1:1000, Cat#DF6967, Affinity), anti-phospho-NF-κB p65 (1:500, Cat#AP0123, ABclonal), anti-NF-κB p65 (1:1000, Cat#A16271, ABclonal), anti-IL-1β (1:1000, Cat#A1112, ABclonal), and anti-β-actin (1:20000, cat#AC026, ABclonal) antibodies. The experiments were independently performed in triplicate.
4
Western Blot Analysis of MAPK Signaling
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A whole‐cell lysis kit (KeyGen) was used to harvest the total protein. Consecutively, 10% SDS‐polyacrylamide gel electrophoresis was performed to separate target proteins, which were then transferred to polyvinylidene fluoride membranes. After blocking in 5% bovine serum albumin for 1 hour, target membranes were incubated with anti‐ GAPDH [6C5] (1:1000; Abcam), JNK (1:1000; Cell Signaling Technology), phospho‐JNK [81E11] (1:1000; CST), P38 [D13E1] (1:1000; CST), phospho‐P38 [D3F9] (1:1000; CST), ERK [137F5] (1:1000; CST), and phospho‐ERK [D13.14.4E] (1:1000; CST) primary antibodies overnight at 4°C. After washing with TBST solution (0.1% Tween‐20, 10 mM Tris‐base, and 100 mM NaCl; pH 7), membranes were incubated with an appropriate secondary antibody (1:1000; Abcam) for 1 hour. Finally, the Bio‐Rad enhanced chemiluminescence detection system was used to detect each protein. Quantification was performed by using the ImageJ software.
5
Exosome Protein Profiling by Western Blot
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Purified exosome pellets were lysed with RIPA lysis buffer (Beyotime, China) and PMSF (Beyotime, China). The cells were harvested and lysed using whole cell lysis kit (Keygen biotech, China). The protein concentrations were determined using a BCA protein assay kit (Thermo Scientific, USA). After boiling, equal amounts of proteins (20 μg) from different samples were separated by 10% SDS polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was then blocked with 5% skim milk for 60 min at room temperature and incubated with different primary antibodies overnight at 4 °C, including anti-CD63 (Abcam, USA), anti-CD9 (Abcam, USA), anti-TSG 101 (Abcam, USA), anti-osterix (Abcam, USA), and anti-Runx2 antibodies (Abcam, USA). The PVDF membrane was further incubated with horseradish peroxidase-tagged secondary antibodies separately for 1 h. Protein bands were visualized using enhanced chemiluminescence assay (ECL, Amersham Biosciences, USA) and imaged by the Bio-RAD ChemiDoc gel imaging system (ChemiDoc XRS + system, Bio-Rad, USA).
6
Western Blot Analysis of SOD2 Protein
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Total proteins were extracted from the above-mentioned cell lines and fresh tissues by using a whole cell lysis kit (Keygen, KGP2100, China). The Western blot procedure is as previously described [47 (link)]. Briefly, 30 μg of total protein from each sample was separated in 12% SDS-PAGE gel and transferred to PVDF membrane (Millipore, Billerica, MA). After blocking in PBS buffer containing 6% nonfat milk at room temperature for 1 hr, the membrane was incubated with rabbit anti-SOD2 (1:5000, Cat No. ab13533, Abcam, USA) and anti-GAPDH (1:5000, Cat No. 5174, Cell Signaling Technology, USA) primary antibodies at 4°C overnight. After washing with TBS buffer containing 0.1% Triton X-100 for three times, the membrane was then probed with anti-rabbit HRP-conjugated secondary antibodies (dilution for 1:5000, Jackson Immunoresearch Inc, PA, USA) and detected by using the enhanced chemiluminescence substrates (PerkinElmer, MA, USA). GAPDH was used as a loading control. The intensity of immunoblot signals was determined using the Bio-Rad software Quantity One (Bio-Rad Laboratories Inc., Hercules, California, USA).
7
Western Blot Analysis of ICP34.5
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A total of 5×106 Vero cells were mock treated or treated with viruses at an MOI (multiplicity of infection) of 2 for 24 h, and then cells were lysed using a Whole Cell Lysis kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). A total of 30 µg protein (quantified using the BCA protein quantification kit; Nanjing KeyGen Biotech Co., Ltd.) was separated by 15% SDS-PAGE and transferred to a 0.22-µm thick nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat dry milk in TBS-Tween-20 (TBS-T, 0.1% Tween) for 1 h at room temperature, the membrane was incubated overnight at 4°C with primary antibodies against ICP34.5 (amino terminus) (diluted 1:500, rabbit polyclonal; cat no. HX5191; TSZ Biosciences, San Fransisco, CA, USA; http://www.tsz-biological.com/ ) or β-actin (diluted 1:1,000; rabbit monoclonal; cat no. 4970; Cell Signaling Technology, Inc., Danvers, MA, USA). The membrane was then washed and blotted with a horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody (diluted 1:6,000; cat no. 7074; Cell Signaling Technology, USA) for 1 h at room temperature. The Protein antibody complexes were visualized with the chemiluminescent HRP substrate (EMD Millipore) using a chemiluminescence imaging system (Tanon-5200; Tanon, Guangzhou, China).
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