RPTCs were treated with or without 20 μmol/L cisplatin for 24 hours. Total cell proteins were extracted using SDS lysis buffer containing 1× Protease Inhibitor Cocktail (RF232581; Thermo) and were quantified using BCA Protein Quantification Kit (CW0014; Cwbiotech Company, China). Kidney cortex tissues were homogenized in SDS lysis buffer containing 1× Protease Inhibitor Cocktail at 4°C and then were centrifuged at 15 322 g for 5 minutes at 4°C. The supernatants were used for protein quantification. Protein samples were separated using SDS‐PAGE (polyacrylamide gel electrophoresis) and then were transferred onto PVDF membrane (IPVH00010; Immobilon) hydrated with methanol. The membranes were incubated with primary antibodies over night at 4°C and secondary antibodies for 2 hours at room temperature. The primary antibodies were rabbit anti‐HNF1β antibody (12533‐1‐AP; Thermo Fisher Scientific), rabbit anti‐caspase 3 antibody (9662; CST) and rabbit anti‐cleaved PARP antibody (BS7047; BioWord) purchased from Cell Signaling Company. The second antibodies were HRP‐labelled anti‐mouse (AS003; ABdone) or anti‐rabbit IgG antibody (AS014; ABdone). The ECL Chemiluminescence Detection Kit (WBKLS0500; Millipore Company) was used for signal detection. The protein bands were quantified using ImageJ software.
Bca protein quantification kit
Manufactured by CWBIO
Sourced in China
The BCA protein quantification kit is a laboratory tool used to determine the concentration of proteins in a sample. It employs the bicinchoninic acid (BCA) assay, a colorimetric detection method that measures the reduction of copper ions by proteins in an alkaline environment. The resulting purple-colored reaction product can be measured spectrophotometrically, allowing for the quantification of protein levels.
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Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ni nta agarose column, by GE Healthcare (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Anti vegf a, by Santa Cruz Biotechnology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Maxima sybr green qpcr master mix 2, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Centrifuge, by Merck Group (1 mentions)
Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit or anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Ecl western blot kit, by Beyotime (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
Goat anti rabbit igg zb 2301, by ZSGB-BIO (1 mentions)
11 protocols using bca protein quantification kit
1
Quantification of Kidney Protein Expression
2
Purification of a Recombinant Protein
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Following induction using methanol for 96 h, the supernatant of the high-expressing clone was collected and dialyzed against a binding buffer (20 mM phosphate buffer, 300 mM NaCl, pH 7.4) overnight using a 10 kDa cutoff dialysis membrane (Millipore, Billerica, MA, USA). Subsequent to filtering through 0.22 um filters (Millipore, Billerica, MA, USA), the supernatants were added to a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (GE, Boston, FA, USA) with 1 mL/min flow velocity, and then the column was washed with ten column volumes of binding buffer. The interest protein bound to the column was eluted with 250 mM imidazole in a binding buffer. The elution fraction containing the interest protein was dialyzed against PBS overnight to remove the imidazole. Finally, the protein was concentrated using ultrafiltration and the concentration of the purified protein was measured using a BCA protein quantification kit (CW biotech, Beijing, China) and HPLC (Shimadzu, Tyoto, Japan) was used to determine the purity of this BsDb, as described in our previous study [47 (link)].
3
Western Blot Analysis of VEGFA Protein
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HEPM and HEK-293 cells were collected after transfection for 48 hours. RIPA lysis buffer (Cwbiotech, China) was used to extract total proteins and proteins were quantified with a BCA protein quantification kit (Cwbiotech, China). 30 μg total protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Then, protein samples on the PVDF membranes were incubated with a primary anti-VEGFA (Santa Cruz, USA) or anti-GAPDH (Cell Signaling Technology, USA) antibody at 4 °C for 12 hours and then with secondary antibodies at room temperature for 2 hours. Finally, proteins were detected with the Odyssey® LI‐COR Imaging System (LI‐COR Biotechnology, USA).
4
Liver Injury Biomarker Analysis Protocol
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Hydroxyproline, alanine transaminase (ALT) and aspartate aminotransferase (AST) testing kits were purchased from Nanjing Jiancheng Co., Ltd., China. PCR primers were purchased from Invitrogen. Maxima SYBR Green qPCR Master Mix (2×) and the First Strand cDNA Synthesis Kit were purchased from Thermo Fisher Scientific, Inc. The bicinchoninic acid (BCA) Protein Quantification Kit was purchased from CWBIO (China). Primary antibodies against α-SMA and collagen I were purchased from Abcam (UK), and that against DAPDH was purchased from Cell Signaling Technology (USA).
5
Quantifying Chicken Secretory IgA in Ileal Mucosa
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After collecting ileal mucosa samples from broilers, the tissue homogenate was prepared using a ratio of mucosal sample to phosphate buffer saline of 1:9. The homogenate was then centrifuged at 3,000 r/min and 4 °C for 15 min to obtain the supernatant for further use. The chicken secretory IgA (sIgA) level was measured using the chicken sIgA ELISA kit (ml002778, Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). Following the provided protocol, total protein levels were measured using the BCA protein quantification kit (Cwbio, Beijing, China). The sIgA values were expressed as the level of sIgA per gram of protein.
6
Serum Immunoglobulin and Mucosal Factors
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The levels of complement 3 (C3 ), β-defensin, lysozyme (LYZ ), IL-4, IL-1β, and interferon (IFN )-γ in the serum, along with the sIgA content of ileum mucosa were determined according to the manufacturer's instruction (Jiancheng Bioengineering Institute, Nanjing, China). The concentration of serum IgA, IgG, and IgM were measured using the Elisa kits strictly following manufacturer's protocols (Solarbio Bioengineering Institute, Beijing, China). The BCA protein quantification kit (Cwbio, Beijing, China) was used to evaluate the total protein concentration of ileal supernatant according to the manufacturer's instructions.
7
Immunoblotting Analysis of Autophagy Markers
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Immunoblotting was performed as previously described32 (link). Briefly, normal and glioblastoma tissues of mice were cut into small pieces and the total proteins were extracted in RIPA lysis buffer (Applygen, Beijing, China) with the addition of a protease/phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) by sonication for a total of 30 s (3 times, 10 s per time) at 4 °C. The tissue lysate was centrifuged using a centrifuge (Sigma–Aldrich, St. Louis, MO, USA) at 12,500 rpm for 20 min at 4 °C and protein concentrations were measured using a BCA protein quantification kit (CWBIO, Beijing, China). Proteins were separated by electrophoresis on 8% or 10% SDS-PAGE gels and transferred to a 0.45 μm PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% fat-free milk in TBST for 1 h, membranes were immunoblotted with primary antibodies to ATG3, ATG5, ATG7, ATG12, ATG16, P62, LC3B, P53, p-ERK1/2, ERK1/2, p-AMPK, AMPK, p-mTOR, m-TOR (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Proteintech, Rosemont, IL, USA) at the appropriate dilutions with gentle shaking overnight at 4 °C. Blots were incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:2000; Cell Signaling Technology) and bands were visualized using an enhanced chemiluminescence, ECL Western blot kit (Beyotime Biotechnology, Shanghai, China).
8
Cell Cycle and Apoptosis Analysis
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Cell cycle staining kit (CCS012) and Annexin V-FITC/PI apoptosis kit (AP101-100-kit) were purchased from Multi-Sciences, Beijing, China. Trizon Reagent (CW0580S), ultrapure RNA extraction kit (CW0581M), HiFiScript first strand cDNA synthesis kit (CW2569M), miRNA purification and reverse transcription kit (CW0627S), ultraSYBR mixture (CW0957M) and BCA protein quantification kit (CW0014S) were products of CWBIO, Beijing, China. Ultrasensitive luminescence solution (RJ239676) was from Thermo Fisher, USA. Mouse monoclonal antibody against GAPDH (TA-08, 1:1,000), goat anti rabbit IgG (ZB-2301, 1:500) and goat anti mouse IgG (ZB-2305, 1:500) were purchased from Zsbio, Beijing, China. Mouse monoclonal antibody against P53 (cat no. 2524) was obtained from Cell Signaling, USA. Rabbit monoclonal antibody against Bax (ab32503) and polyclonal antibody against Bcl-2 were from Abcam, UK and Bioss, USA, respectively.
Flow cytometer (NovoCyteTM) was from Eisen Biologicals, Hangzhou, China. Fluorescent PCR instrument (CFX Connect™) and ultrasensitive chemiluminescence imaging system (Chemi DocTM XRS+) were purchased from Bio-Rad, Shanghai, China.
Flow cytometer (NovoCyteTM) was from Eisen Biologicals, Hangzhou, China. Fluorescent PCR instrument (CFX Connect™) and ultrasensitive chemiluminescence imaging system (Chemi DocTM XRS+) were purchased from Bio-Rad, Shanghai, China.
9
Protein Extraction and Analysis
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The cells were cultured in 25 cm2 flasks. Molding was performed 24 h after drug administration, and then the proteins were extracted and purified using the mammalian protein extraction kit (CW Biotech, Beijing, China). Then, the proteins were quantified by the BCA protein quantification kit (CW Biotech, Beijing, China). Protein extracts were diluted to the same concentration and added into the loading buffer (CW Biotech, Beijing, China) at a ratio of 1 : 4 for 10 min in a boiling water bath. The protein samples were separated from the sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto the polyvinylidene difluoride (PVDF) membrane using a semidry blotter. The resulting PVDF membrane was blocked for 2 h using the blocking buffer [5% milk in Tris-buffered saline with Tween 20 (TBST)]. Then, primary antibodies were prepared and incubated overnight at 4 °C. The membrane was washed three times using TBST. The membrane was incubated with secondary antibodies in the blocking buffer for 2 h and then washed three times using TBST. After adding the enhanced chemiluminescence, the membrane was incubated in the dark for 5 min. The band intensities were quantified using the Photo-Image System (Molecular Dynamics, Sweden).
10
Purification of recombinant PyasOBP2 protein
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The culture was diluted 1:100 with liquid LB, and incubated at 37°C until the OD600 reached a value of 0.6–0.8. Protein expression was induced by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM into the culture, and allowed to last for 10 h at 18°C. The bacterial cells (500 ml) were collected by centrifugation (8000 g for 10 min, 4°C), and the cell pellet was suspended with the lysis buffer (50 mg/ml Lysozyme and 20 mM Tris-HCl buffer at pH 7.4). The suspension was sonicated on ice, and centrifuged (12000 g for 30 min, 4°C) for a second time. Recombinant PyasOBP2 was examined by Sodium Dodecyl Sulfate—Polyacrylamide Gel electrophoresis (SDS-PAGE). Protein present in the supernatant was purified with a Ni-NTA His·Bind Resin column (7 Sea Biotech, Shanghai, China). The purified protein was assessed by SDS-PAGE, identified with the anti-His tag monoclonal antibody (Cwbio biotech, Beijing, China) by the Western Blot analysis, and desalted in a dialysis buffer (20 mM Tris-HCl at pH 7.4). To avoid confounding effects on subsequent experiments, His-tag was removed from the protein using a recombinant enterokinase (rEK) (Yeasen Biotech, Shanghai, China), and the concentration of the protein was assayed by the BCA protein quantification kit (Cwbio biotech, Beijing, China).
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