HeLa cells that transfected with plasmid DNA encoding different spike mutants using Lipofectamine 3000 were seeded on glass coverslips 48 h after transfection in a humidified incubator at 37 °C with 5% CO2. Twenty-four hours after seeding, the cells were washed with PBS, fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated in QuickBlock buffer (Beyotime) for 10 min. The cells were washed and incubated with a rabbit polyclonal antibody against spike protein (1:400) for 1 h, followed by staining with AF555-conjugated goat anti-rabbit IgG (1:500) to detect the cell-surface spike protein. Cells were subsequently washed five times in PBS and permeabilized with Triton, followed by incubation in QuickBlock buffer (Beyotime) for 10 min. Then, the cells were washed and incubated with a rabbit polyclonal antibody against spike protein (1:400) for 1 h, followed by staining with AF488-conjugated goat anti-rabbit IgG (1:500) to label the intracellular spike protein (as well as available cell-surface S protein). After counterstaining the nuclei with DAPI, the cells were mounted in Dako Fluorescent Mounting Medium (S3023) and imaged using a Leica TCS SP8 confocal microscope equipped with Leica Application Suite X software.
Quickblock buffer
Manufactured by Beyotime
Sourced in China
QuickBlock buffer is a reagent used in the extraction of proteins from biological samples. It serves as a lysis buffer, facilitating the rapid and efficient disruption of cell membranes and the release of intracellular proteins. The buffer composition is designed to maintain the stability and solubility of the extracted proteins, making it suitable for various protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
F actin, by Yeasen (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Triton x 100, by Beyotime (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti ib4, by Merck Group (1 mentions)
Mouse anti cgrp, by Abcam (1 mentions)
Goat anti gfap, by Abcam (1 mentions)
Mouse anti ox 42, by Abcam (1 mentions)
Alexa 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated donkey anti goat igg, by Jackson ImmunoResearch (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Cryostat, by Leica camera (1 mentions)
Spectra multicolour high range protein ladder, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Ripa solution, by Beyotime (1 mentions)
23 protocols using quickblock buffer
1
Visualizing SARS-CoV-2 Spike Protein Expression
2
Immunohistochemical Analysis of Rat Spinal Cord
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Rats were deeply anesthetized with 0.4% sodium pentobarbital anesthesia (40 mg/kg
body weight, i.p.) and perfused transcardially with 500 ml of cold
phosphate-buffered saline (PBS) followed by 500 ml of cold 4% paraformaldehyde
(PFA). The spinal cord was removed and postfixed with the same 4% PFA for 1–2 h
at 4°C and then transferred to 30% sucrose in PBS overnight. Sample sections (20
μm thickness) were adhered on gelatin-coated glass slide with a cryostat
(Leica). The sections were washed three times with PBS pH = 7.35–7.40 and then
hatched in PBST (0.3% Triton in PBS) for 40 min, blocked with QuickBlock buffer
(Beyotime, P0260) for 10 min, subsequently incubated overnight at 4°C with
primary antibody for rabbit-anti-p-PKC (1:200, Abcam),
mouse-anti-CGRP (1:200, Abcam), goat-anti-GFAP (1:200, Abcam),
mouse-anti-OX-42(1:200, Abcam), anti-IB4(1:50, Sigma), and mouse-anti-NeuN
(1:200, Millipore). The sections were then incubated for 60 min at room
temperature with secondary antibodies Cy3-conjugated donkey anti-rabbit IgG
(1:200, Jackson ImmunoResearch), FITC-conjugated donkey anti-goat IgG (1:200,
Jackson ImmunoResearch), and Alexa 488-conjugated donkey anti-mouse IgG (1:200,
Thermofisher). The stained sections were captured with LSM 780 (Carl Zeiss). The
fluorescent density was quantified with a computer-assisted imaging analysis
system (ImageJ, National Institutes of Health).
body weight, i.p.) and perfused transcardially with 500 ml of cold
phosphate-buffered saline (PBS) followed by 500 ml of cold 4% paraformaldehyde
(PFA). The spinal cord was removed and postfixed with the same 4% PFA for 1–2 h
at 4°C and then transferred to 30% sucrose in PBS overnight. Sample sections (20
μm thickness) were adhered on gelatin-coated glass slide with a cryostat
(Leica). The sections were washed three times with PBS pH = 7.35–7.40 and then
hatched in PBST (0.3% Triton in PBS) for 40 min, blocked with QuickBlock buffer
(Beyotime, P0260) for 10 min, subsequently incubated overnight at 4°C with
primary antibody for rabbit-anti-p-PKC (1:200, Abcam),
mouse-anti-CGRP (1:200, Abcam), goat-anti-GFAP (1:200, Abcam),
mouse-anti-OX-42(1:200, Abcam), anti-IB4(1:50, Sigma), and mouse-anti-NeuN
(1:200, Millipore). The sections were then incubated for 60 min at room
temperature with secondary antibodies Cy3-conjugated donkey anti-rabbit IgG
(1:200, Jackson ImmunoResearch), FITC-conjugated donkey anti-goat IgG (1:200,
Jackson ImmunoResearch), and Alexa 488-conjugated donkey anti-mouse IgG (1:200,
Thermofisher). The stained sections were captured with LSM 780 (Carl Zeiss). The
fluorescent density was quantified with a computer-assisted imaging analysis
system (ImageJ, National Institutes of Health).
3
Western Blot Protein Analysis from HUVECs
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Protein was extracted from HUVECs by RIPA solution (P0013B, Beyotime) combined with phenylmethanesulfonyl fluoride (PMSF) and a phosphatase inhibitor. Then, all the protein samples were mixed with 1X SDS–PAGE protein loading buffer (P0015A, Beyotime). After all the samples were denatured at 100°C for 10 min, electrophoresis was performed using a 10% SDS–PAGE gel preparation (Bio-Rad, USA) at 80 V for 30 min and 120 V for 90 min. Then, the proteins were blotted onto a di-fluoride polyvinylidene fluoride (PVDF) membrane at 240 mA for 2 h in ice water. Next, the PVDF membrane was blocked in QuickBlock™ buffer (P0252, Beyotime) for 40 min and then incubated with diluted primary antibodies (Table 3 ) at 4°C overnight. After washing three times with tris buffered saline with tween (TBST), horseradish-peroxidase-conjugated (HRP-conjugated) antibodies were added to the PVDF membrane. After three washes, the immunoreactive bands were visualized using ChemiDoc Touch (Bio-Rad, USA). We used a Spectra multicolour high-range protein ladder (26 625, Thermo Scientific) as the protein ladder.
4
Western Blot Analysis of Ovarian Proteins
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One ovary (approximately 40 mg) was ground in a KZ‐II High‐throughput tissue grinder (Servicebio), after which the total protein was extracted. The well‐boiled protein samples were separated by 10% or 12% BeyoGel™ Plus Precast Tris‐Gly PAGE Gel (Beyotime). Then, they were blocked with QuickBlock™ buffer (Beyotime) for 15 min, incubated with primary antibodies (collagen I, p‐Smad2, Smad2, CTGF, TGF‐β1, p‐Smad3, Smad3, PPAR‐γ, β‐actin, Smad4, α‐SMA, and Smad7) for 3.5 h at 4°C, and then incubated with antirabbit IgG for 90 min at 37°C. Subsequently, all bands were observed in the FluorChem M System (ProteinSimple, Santa Clara, CA, USA) after reaction with the Enhanced Chemiluminescence (ECL) detection kit, and bands were analyzed for grayscale using ImageJ (https://imagej.nih.gov/ij/ , NIH Image, Bethesda, MD, USA).
5
Immunohistochemical Analysis of BMPR2 Expression
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The paraffin-embedded sections were heated in citrate buffer (pH 6.0) at 95°C for 15 min for antigen retrieval. The sections were then incubated in 3% H2O2 for 20 min and were blocked using 10% goat serum (Sigma-Aldrich, Saint-Louis, USA) for 1 h to quench the endogenous peroxidase activity. They were then incubated with a primary antibody against BMPR2 (#ab130206, 1:200, Abcam, Cambridge, MA, USA) for 1.5 h and with HRP-conjugated IgG (#ab64264, 1:500, Abcam) for another 1 h. The primary antibody was diluted in a QuickBlock buffer (#P0262, Beyotime, Shanghai, China), and buffer without antibody was used as a negative control. The immunorections were developed using DAB reagent (Beyotime Institute of Biotechnology, Jiangsu, China) and were counterstained with hematoxylin (Beyotime). The tissue sections were analyzed and photographed with a microscope (BX60; Olympus, Tokyo, Japan).
6
Protein Expression Analysis in Cultured mDPCs
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Cultured mDPCs were rinsed with PBS twice and lysed in radioimmunoprecipitation assay (RIPA) buffer followed by centrifugation at 16,000 g at 4 °C for 10 min. The concentration of total protein in the supernatant was measured using the BCA Protein Assay Kit (Pierce Biotechnology, IL, USA). Equal amounts of protein were loaded and separated on an 8% polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Roche). The membranes were then blocked in QuickBlock™ buffer (Beyotime Biotechnology) at room temperature for 15 min, followed by incubation with the primary antibodies. Then, membranes were incubated with secondary antibodies at room temperature for 1 h, visualized by SuperSignal West Pico PLUS (Thermo Fisher) reagents and imaged by an Odyssey CLx Imaging System (LI-COR, NE, USA).
7
Western Blot Analysis of HGF Proteins
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HGFs were cultured onto each sample in 24-well plates at a density of 5 × 104 cells per well with or without CM for Western blot experiments. After 7 days of culture, HGFs were lysed in RIPA buffer and proteins were separated on 8% SDS-PAGE gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes which were blocked for 1 h by QuickBlock buffer (Beyotime, Jiangsu, China) at room temperature. Membranes were washed in TBST and probed overnight at 4°C with one of the following primary antibodies: collagen-I (ab34710, 1:2,000; Abcam, USA), vinculin (ab129002, 1:10,000; Abcam, USA), fibronectin (ab2413, 1:1,000; Abcam, USA), and β-actin (1:1,000, ab8227; Abcam, USA). Membranes were washed in TBST and incubated for 1 h with HRP-conjugated secondary antibodies at room temperature. Proteins were visualized using the ECL system (Beyotime, Jiangsu, China).
8
Western Blot Analysis of Inflammatory Mediators
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Cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing proteinase and phosphatase inhibitors. A BCA assay kit (Beyotime) was used to measure the concentration of protein. The supernatant proteins were precipitated. The protein of lytic samples was transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, China) after separating by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime). Membranes were incubated with antibodies against gasdermin D (GSDMD, Abcam, Cambridge, UK), IL-1β (Abcam), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc-1, Abcam), TRAP (Abcam), pro caspase-1 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-1 (Cell Signaling Technology), ASC (Cell Signaling Technology), TNF receptor-associated factor (TRAF) 2 (Proteintech, Rosemont, IL, USA), TRAF6 (Proteintech), c-Fos (Proteintech), cathepsin K (CTSK, Proteintech), matrix metalloprotein 9 (MMP9, Proteintech), and anti-actin (Beyotime) overnight at 4 °C after blocking with quick block buffer (Beyotime) for 30 min. Then, membranes were washed three times with TBS-Tween and incubated with the appropriate secondary antibody for 1 h at room temperature. The results were visualised via chemiluminescent peroxidase substrate (Proteintech).
9
Mitochondrial Morphology Analysis in HL-1 Cells
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To observe the mitochondrial morphology, HL-1 cells were stained with a mitochondria-specific Tom-20 (translocase of outer mitochondria 20) antibody, following a method that has been widely described previously [28 (link), 29 ] and used in our laboratory [30 ]. Briefly, HL-1 cells were fixed with 4% paraformaldehyde (PFA), permeabilized with Triton X-100, and blocked with a QuickBlock buffer (Beyotime). The primary antibody against Tom-20 (Cell Signaling Technology, Danvers, MA, USA) was applied overnight at 4 °C, followed by incubation with a CoraLite594 conjugated secondary antibody (Proteintech Group, Rosemont, IL, USA). Nuclei were stained with a DAPI solution (Beyotime) for 5 min.The stained cells were imaged with a 63 × oil objective on the ZEISS confocal microscopy. Image analysis was performed using Mito-morphology macro in the ImageJ software, based on the methods described by Fonseca et al. [28 (link)]. Mitochondrial size was assessed by the average area and perimeter, while shape was gauged by circularity. Fragmented and spherical mitochondria, indicative of fission, were identified by decreased size and increased circularity [28 (link), 29 ].
10
Immunofluorescence Staining of Fibroblast-Like Synoviocytes
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After treating FLSs with various interventions, the cells were fixed using 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Beyotime, China) on ice. Then, cells were blocked with QuickBlock buffer (Beyotime, China) for another hour. The cells were incubated primary antibodies against ADAM8, IL-6, and TNF-α protected from light at 4 °C overnight, followed by incubation with F-actin (Yeasen, China). To stain cell nuclei, DAPI (Beyotime, China) was used for a duration of 10 min. The FLSs were then observed and photographed using a Zeiss laser scanning microscope (LSM 510, Zeiss).
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