At cryogenic temperatures, the minced lung was homogenized in saline by an Ultra-Turrax homogenizer (IKA, Staufen, Germany). The supernatant was harvested for the next step, after the homogenate of lung tissue was centrifuged for 15 min at 3000 rpm. Commercial kits (Jiancheng, Nanjing, China) were used to measure redox indicators. All protocols were conducted as described in the manufacturer’s instructions. All of the kits this study used in this section are listed below: GSH (glutathione) concentration, CAT, H2O2 concentration, TrxR, SOD, MDA (malondialdehyde) concentration, and T-AOC (total antioxidant capacity). ELISA kits from the Jingmei Institute of Biotechnology (Jingmei, Wuxi, China) were used to detect cytokine levels. Microplate readers (BioTek Epoch, Winooski, VT, USA) were used to measure the absorbance. The standard curve R2 value was >0.990, the inter-plate and intra-plate variabilities were <15%. The BCA kits (Meilunbio, Dalian, China) were used to assess protein concentrations, which were used to standardize all the data in this section.
Bca kit
Manufactured by Meilun
Sourced in China
The BCA kit is a colorimetric assay used for the quantitative determination of total protein concentration. It utilizes bicinchoninic acid (BCA) as the detection reagent, which forms a purple-colored complex with proteins in an alkaline environment. The intensity of the color is proportional to the protein concentration, allowing for accurate protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Meilun (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Tbst buffer, by Meilun (2 mentions)
Dimethyl sulfoxide (dmso), by Maclin Biochemical Technology (1 mentions)
Pink1, by ABclonal (1 mentions)
Parkin, by ABclonal (1 mentions)
Tom20, by ABclonal (1 mentions)
Loading buffer, by Meilun (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Dulbecco modified eagle medium (dmem), by Meilun (1 mentions)
Fetal bovine serum (fbs), by Meilun (1 mentions)
Penicillin streptomycin, by Meilun (1 mentions)
β tubulin, by ABclonal (1 mentions)
Mda kit, by Solarbio (1 mentions)
Phosphatase inhibitor cocktail, by AbMole (1 mentions)
Protease inhibitor cocktail, by AbMole (1 mentions)
Vegfa, by ABclonal (1 mentions)
Hif 1α, by ABclonal (1 mentions)
β actin, by ABclonal (1 mentions)
14 protocols using bca kit
1
Antioxidant and Cytokine Analysis of Cryogenic Lung Tissue
2
Cardioprotective Effects of Natural Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following reagents were used: DMEM, FBS, penicillin/streptomycin (Meilunbio, Dalian, China), cTnI Elisa kits (Sangon Biotech, Shanghai, China), TNF-α, IL-1β, and IL-6 Elisa kits (MyBioSource, San Diego, United States), MDA kits, SOD kits, and NADH kits (Solarbio, Beijing, China), CCK-8 kits (Beyotime Biotechnology, Shanghai, China), ROS fluorimetric kits, JC-1 fluorescent dye, protein extraction kits, and BCA kits (Meilunbio, Dalian, China). IL-1 (1:100), PINK1(1:1,000), Parkin (1:1,000), AMPK(1:1,000), Akt (1:1,000), P13K(1:1,000), Tom20 (1:1,000), β-tubulin (1:1,000), mTOR (1:1,000), and an HRP-conjugated GAPDH (1:1,000) antibody were obtained from Abclonal Biotechnology (Wuhan, China). The RIPA lysis buffer and loading buffer were purchased from Meilunbio (Dalian, China). PVDF membranes were obtained from Merck (New Jersey, United States). DMSO was purchased from Macklin (Shanghai, China), and doxorubicin, breviscapine, and dexrazoxane were purchased from Widely (Wuhan, China). The Mdivi-1 inhibitor was purchased from Selleck Chemicals (Houston, United States).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from theca layer and granulosa layer using RIPA Lysis Buffer (Beyotime, Shanghai, China; P0013B) and Protease Inhibitor Cocktail (AbMole, Shanghai, China; M5293), Phosphatase Inhibitor Cocktail (AbMole, Shanghai, China; M7528). Total protein content was determined using a BCA kit (Meilunbio, Dalian, China; MA0082). Proteins were transferred to Polyvinylidene fluoride membranes following migration through SDS-PAGE gels of gradient concentrations. Following blocking with skimmed milk powder, membranes were incubated with primary antibodies to protein to detect CD31 (1:1000, ABclonal, Wuhan, China; A0378), VEGFA (1:200, ABclonal, Wuhan, China; A12303), VEGFR (1:500, ABclonal, Wuhan, China; A11127), P-VEGFR (1:500, ABclonal, Wuhan, China; AP0382), HIF1α (1:1000, ABclonal, Wuhan, China; A0378), β-actin (1:2000, ABclonal, Wuhan, China; AC038). Secondary antibodies were combined with primary antibodies and protein bands were visualized with a chemiluminescence system (Tanon, Shanghai, China) and quantified with Image J software.
4
Western Blot Analysis of Hippocampal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein concentration in the hippocampus or cells was detected using a BCA kit (Meilunbio, Dalian, China). Briefly, the total protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Following the blocking step with 5% nonfat milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with specific primary antibodies, namely β-actin (1:2000) (Elabscience Biotechnology, Wuhan, China), ALOX15 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nrf2 (1:500), HO-1 (1:500), VDR (1:1000), GPX4 (1:1000), NeuN (1:1000), and ACSL4 (1:500) (Affinity, Changzhou, China). After washing by TBST, the membranes were incubated with peroxidase-conjugated goat anti-rabat or goat anti-mouse IgG (EarthOx, San Francisco, CA, USA) for 1 h at room temperature. Then, the protein bands were visualized using Pierce ECL Western blotting Substrate (Engreen Biosystem, Beijing, China). The relative optical density values of protein bands were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
5
Quantifying Ferrous Iron in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An iron content detection kit (Solarbio, China) was used to measure the content of Fe2+ according to the manufacturer’s instructions. Briefly, the treated HEI-OC1 cells or cochlear tissues were lysed with extract and centrifuged at 4,000 × g for 10 min at 4°C. The supernatant was collected and boiled for 5 min after being mixed with detection reagent. Then chloroform was added to the mixture and centrifuged at 10,000 × rpm for 10 min at room temperature. The newly collected supernatant was added to a 96-well plate, and then the absorbance of the samples at 520 nm was recorded using a SpectraMax i3x (Molecular Devices, USA). The protein concentration of the samples was quantified by a BCA kit (Meilunbio, China). Relative Fe2+ level was expressed as μg/mg protein.
6
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were first harvested from the culture medium. Subsequent protein extraction was performed on ice for 30 min using RIPA lysis buffer (P0013B; Beyotime, Beijing, China) and protease and phosphatase inhibitor cocktail (P1046; Beyotime). The protein sample was at 13 400 g for 30 min at 4 °C. The proteins were concentrated using a BCA kit (MA0082, Meilunbio, Dalian, China), and western blotting was performed as previously described. Primary antibodies against DNMT1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The β‐actin was purchased from Affinity Biosciences (Affinity, Cincinnati, OH, USA). The goat anti‐rabbit IgG and anti‐mouse IgG secondary antibodies were purchased from Affinity Biosciences (Affinity).
7
Gelatin Zymography Protocol for Protease Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein concentration was measured using a BCA kit (Meilunbio, Shanghai, China). The extracted protein (60 mg) with 4 × Zymography Sample Buffer in a volume ratio of 3:1 and then loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels mixed with 15% gelatin for electrophoresis. SDS was removed from the gels to renature the protein with 2.5% Triton X-100 three times for 20 min each. Incubated the gels in 1 × LSCB buffer (10 ×: Ph = 7.6, 0.5 M Tris Base, 2.0 M NaCl, 0.05 M CaCl2, 0.2% (w/v) Brij-35) for 72 h at 37 °C. The gels were incubated with Working Coomassie-G Dye (12.5% Coomassie Brilliant Blue, 10% acetic acid, 50% methanol, 27.5% ddH2O) at 80 rpm for 1 h and washed with decoloring solution (10% acetic acid, 20% methanol, 70% ddH2O). Results were recorded using a scanner (Brother, Shanghai, China).
8
Evaluating Oxidative Stress in Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DCFH-DA (Biosharp Co., Ltd., Beijing, China) and flow cytometry were used to detect changes in intracellular ROS levels. Briefly, fresh brain tissues were prepared in a single-cell suspension and then incubated with 10 μM H2DCFDA. The cells were washed and collected for fluorescence detection by flow cytometry.
CAT, SOD, GSH-Px, MDA, IL-1β, TNF-α, and IL-6 levels in the brain and Serum LPS levels in the serum were analyzed with the corresponding kits (Boster, Wuhan, China). The brain tissues were prepared as 10% tissue homogenate in a homogenizer and protein was quantified with a BCA kit (Dalian Meilun Biological Technology Co., Ltd., Dalian, China) [34 (link)].
CAT, SOD, GSH-Px, MDA, IL-1β, TNF-α, and IL-6 levels in the brain and Serum LPS levels in the serum were analyzed with the corresponding kits (Boster, Wuhan, China). The brain tissues were prepared as 10% tissue homogenate in a homogenizer and protein was quantified with a BCA kit (Dalian Meilun Biological Technology Co., Ltd., Dalian, China) [34 (link)].
9
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed on ice for 30 minutes in RIPA lysis buffer (Meilunbio, China) containing phenylmethanesulfonyl fluoride (0.1 mg/mL). The supernatant was centrifuged to obtain the sample of the protein. The protein was quantified by a BCA kit (Meilunbio, China) with 10 μL of the supernatant taken. 40μg protein sample was loaded to 8% -12% gel electrophoresis SDS-PAGE. The protein was transferred to the polyvinylidene fluoride (PVDF) Membrane (Millipore, USA, ThermoFisher, USA). The PVDF membrane was blocked in a blocking solution by the TBST buffer (Meilunbio, China), which contains 5% skim milk powder (BD, USA) for one hour. The PVDF membrane was incubated with the primary antibody at 4 °C overnight and then incubated with the corresponding secondary antibody for 1 hour at room temperature. After being washed three times with TBST buffer (Meilunbio, China), The PVDF membrane was detected by Western fluorescence assay Beyo ECL Plus (Beyotime, China, Meilunbio, China). The following antibodies were used: Anti-CD44 (1:2000, CST, USA), Anti-EpCAM (1:1000, CST, USA), Anti-MKL-1 (1:1000, CST, USA), Anti-GAPDH (1:1000, Abcam, USA).
10
Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein extraction buffer (Beyotime, China) was used to extract the protein from the tongue cancer tissues and cells. A BCA kit (Meilunbio, China) was used to measure protein concentration. Notably, 10% SDS-PAGE gels were used to isolate proteins with different molecular weights when the protein concentration of the experimental and the control groups was the same. Next, the PVDF membrane was used to transfer the isolated proteins. The PVDF membranes were blocked with 5% non-fat milk powder for 1 h, then incubated with antibodies against E-cadherin (Proteintech, USA) and GAPDH (Bioworld, USA) at 4°C overnight. The PVDF membrane was then washed with TBST and incubated with secondary antibodies (Boster, China) for 2 h. ECL Super Signal (Thermo Fisher Scientific, USA) was used to detect the protein bands. ImageJ software was used for quantitative analysis of protein bands.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!