Following HG stimulation, the HK-2 cells were lysed in lysis buffer (BestBio) and centrifuged (12,000 ×g, 4 ℃) for 20 min to acquire total proteins. The proteins were then transferred to polyvinylidene fluoride membranes after sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The membranes were placed in 5% non-fat milk at room temperature for 1 h before being incubated with the corresponding primary antibodies overnight at 4 ℃, and the goat anti-rabbit horseradish peroxidase secondary antibody (1/2,000; Abcam) at room temperature for 2 h. The signals were developed using the enhanced chemiluminescence reagent (Advansta, Inc.), and the results were measured using ImageJ 1.46 (National Institutes of Health, USA).
Lysis buffer
Manufactured by BestBio
Sourced in China
Lysis buffer is a solution designed to break down and disrupt the cell membranes and walls of biological samples, such as cells or tissues. It is a commonly used reagent in various molecular biology and biochemistry applications to extract and isolate cellular components, such as proteins, nucleic acids, or organelles, for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by BestBio (3 mentions)
Goat anti mouse igg, by ZenBio (3 mentions)
Enhanced chemiluminescence reagent, by Advansta (1 mentions)
Dichlorodihydrofluorescein diacetate, by Merck Group (1 mentions)
Atp bioluminescent assay kit, by Promega (1 mentions)
Odyssey ir imaging system, by LI COR (1 mentions)
Lysis buffer, by Keygen Biotech (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Protease inhibitor cocktail, by BestBio (1 mentions)
Foxo1, by Proteintech (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Ripa buffer, by Leagene (1 mentions)
α sma, by Abcam (1 mentions)
Col 1, by Bioss Antibodies (1 mentions)
Hrp conjugated secondary antibody, by Bioss Antibodies (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Erk1 2, by Abcam (1 mentions)
9 protocols using lysis buffer
1
Western Blot Analysis of Protein Expression
2
ATP and ROS Measurement in HK-2 Cells
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The HK-2 cells were lysed in lysis buffer (BestBio) and centrifuged (12,000 ×g, 4 ℃) for 20 min. Adenosine triphosphate (ATP) activity in the cell supernatant was detected using the ATP Bioluminescent Assay Kit (Promega Corporation) in accordance with the manufacturer’s instructions. To examine intracellular reactive oxygen species (ROS), 10 µΜ of dichloro-dihydro-fluorescein diacetate (Sigma-Aldrich) was used to treat the HK-2 cells at 37 ℃ for half an hour in the dark and the cells were then washed thrice with serum-starved RPMI-1640. The results were photographed with a fluorescence microscope (magnification ×200).
3
Quantification and Immunoblotting of PMCA4
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Uterine tubular gland cells were lysed in lysis buffer (BestBio) and the total protein concentration was quantified using a BCA assay (BestBio) according to the manufacturer’s protocol. Immunoblots were performed using primary and secondary antibodies such as anti-ATP2B4 (PMCA4) (1:1000, Abcam) and goat anti-mouse IgG (Zen-Bio, Chengdu, China) respectively. Western blotting was performed as described previously [71 (link)].
4
Analyzing Cisplatin-Induced Apoptosis
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Transfected or infected HELA or SIHA cells treated with IC50 of cisplatin for 8 hours were lysed for 30 minutes with lysis buffer (Bestbio). Following centrifugation at 11 000 g, supernatants were collected to obtain cytoplasmic proteins and precipitates were collected to obtain mitochondrial proteins. Transfected or infected HELA or SIHA cells treated with IC50 of cisplatin or cells treated with different concentrations of cisplatin for 24 hours were lysed for 30 minutes with lysis buffer (KeyGEN BioTECH) to obtain total protein. Following boiling with loading buffer, protein samples were separated by 10% or 12% SDS‐PAGE and transferred onto PVDF membranes, which were blocked with 5% non‐fat milk for 1 hour. Subsequently, membranes were incubated with primary antibodies at 4°C overnight. Following washing three times, the membranes were incubated with fluorescence‐conjugated secondary antibodies at room temperature in the dark for 1 hour and visualized with Odyssey IR imaging system (LI‐COR Biosciences). Normalization was ensured by α‐tubulin, and each band was quantified using Quantity One software.
5
Western Blot Analysis of Bcl-2 and TLR7
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The protein expression of Bcl-2, TLR7, and related signaling molecules was determined by Western blotting. MFC cells were washed with plain prewarmed phosphate-buffered saline (PBS) after infection. Cells were solubilized in lysis buffer (BestBio, China) and a protease inhibitor cocktail (BestBio, China). Whole cell extracts were mixed in Laemmli loading buffer, boiled for 5 min, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were blocked with 5% non-fat milk for 2 h, transferred onto nitrocellulose membranes, and blotted overnight at 4°C with antibody or anti-β-actin (Santa Cruz Biotechnology, California, USA) (at a dilution of 1: 2000). The membranes were washed with Tris-buffered saline with Tween 20 (TBST) three times and incubated with horseradish peroxidase (HRP)-conjugated second antibody for 1 h. Protein bands were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, USA) and examined with Alpha Ease FC software.
6
Western Blot Analysis of FoxO1 and Akt Signaling
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Total proteins were extracted from mice gastrocnemius muscles and HUVECs using radioimmunoprecipitation (RIPA) buffer (Leagene Biotech, Beijing, China). Nuclear and cytoplasm proteins were extracted from HUVECs in lysis buffer (BestBio, Shanghai, China) following instructions from the manufacturer. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against FoxO1 (proteintech, 1:1000), Phospho-FoxO1-S256 (ABclonal, 1:1000), Akt (cell signaling, 1:1000), Phospho-Akt-S473 (cell signaling, 1:1000), β-actin (cell signaling, 1:1000), GAPDH (Unibo, 1:4000) and Lamin B1 (cell signaling, 1:1000) respectively at 4 °C overnight. On the following day, the membranes were washed and incubated with HRP-conjugated secondary antibodies (anti-rabbit secondary antibodies, cell signaling, 1:2000; anti-mouse secondary antibodies, cell signaling, 1:1000) at 4 °C for an hour. The protein-antibody interaction bands were detected by enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, USA) and visualized by a chemiluminescence detection system (Amersham Imager 600, GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
7
Western Blot Analysis of Liver Proteins
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Liver tissue or JS1 cells were fully lysed with lysis buffer (BestBio, Shanghai, China) at 4°C, total proteins were extracted from lysate suspension, and their concentrations were measured using a BCA protein assay kit. Each protein sample that makes a concentration of 20 μg was electrophoretically separated on 8/15% polyacrylamide gels and then transferred into PVDF membranes (Millipore, Burlington, MA, USA). The membranes were blocked and bound with 5% skim milk for 2 h at room temperature, and probed with antibodies to Col I (1:800) (Bioss, Beijing, China), α-SMA (1:1,000) (Abcam Cambridge, MA), ERK1/2 (1:1,000) (Abcam Cambridge, MA), TGFRB1 (1:1,000) (Proteintech, Chicago, USA), TGFRB2 (1:1,000) (Proteintech, Chicago, USA), and GAPDH (1:2,000) (Abcam Cambridge, MA) at 4°C overnight, and the corresponding HRP-conjugated secondary antibody (Bioss, Beijing, China) was bound for 45 min at room temperature. Then, antigen-antibody reaction was detected by an enhanced chemiluminescence assay (ECL) kit (Millipore, MA, USA). The final results were evaluated with the Image J software, which semiquantitatively estimated the intensity of the grayscale images.
8
Quantitative Protein Analysis in Uterine Cells
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Uterine tubular gland cells were lysed in lysis buffer (BestBio) and the total protein concentration was quantified using a BCA assay (BestBio) according to the manufacturer's protocol.
Immunoblots were performed using prescribed primary and secondary antibodies such as anti-ATP2B4 (PMCA4) (1:1000, Abcam) and goat anti-mouse IgG (Zen-Bio, Chengdu, China) respectively. Western blot procedures were conducted as previously described [67] .
Immunoblots were performed using prescribed primary and secondary antibodies such as anti-ATP2B4 (PMCA4) (1:1000, Abcam) and goat anti-mouse IgG (Zen-Bio, Chengdu, China) respectively. Western blot procedures were conducted as previously described [67] .
9
Quantitative Protein Analysis in Uterine Cells
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Uterine tubular gland cells were lysed in lysis buffer (BestBio) and the total protein concentration was quantified using a BCA assay (BestBio) according to the manufacturer's protocol.
Immunoblots were performed using prescribed primary and secondary antibodies such as anti-ATP2B4 (PMCA4) (1:1000, Abcam) and goat anti-mouse IgG (Zen-Bio, Chengdu, China) respectively. Western blot procedures were conducted as previously described [67] .
Immunoblots were performed using prescribed primary and secondary antibodies such as anti-ATP2B4 (PMCA4) (1:1000, Abcam) and goat anti-mouse IgG (Zen-Bio, Chengdu, China) respectively. Western blot procedures were conducted as previously described [67] .
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