Total proteins were obtained by treating the SC-β cells in RIPA lysis buffer. To obtain equal amounts of protein, the bicinchoninic acid (BCA) protein quantification kit (YEASEN, #20201ES76) was used to measure the protein concentrations. The proteins were separated on 10% SDS/PAGE gels and transferred to nitrocellulose membranes (PALL, #66485). The blots were visualized by enhanced chemiluminescence (ECL) and exposed to an Amersham Imager 600. The uncropped and unprocessed scans of the blots were in the Source Data file.
Bca protein quantification kit
Manufactured by Yeasen
Sourced in China
The BCA Protein Quantification Kit is a colorimetric assay used for the determination of protein concentration. It utilizes the bicinchoninic acid (BCA) method to produce a purple-colored reaction that can be measured spectrophotometrically. The kit provides a simple, reliable, and accurate way to quantify total protein levels in a variety of samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (11 mentions)
Ripa lysis buffer, by Yeasen (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Cell lysis buffer, by Cell Signaling Technology (4 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Page gel quick preparation kit, by Yeasen (2 mentions)
Protease inhibitor cocktail tablet, by Roche (2 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab8227, by Abcam (2 mentions)
Enhanced ecl chemiluminescent substrate kit, by Yeasen (2 mentions)
Ripa lysis buffer strong, by Yeasen (2 mentions)
β actin, by ABclonal (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Super ecl detection reagent, by Yeasen (2 mentions)
Amersham imager 600 system, by GE Healthcare (2 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
71 protocols using bca protein quantification kit
1
Quantitative Western Blot Analysis
2
Protein Extraction and Western Blot Analysis
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Cell and tissue proteins were extracted using lysis buffer for WB/IP assays (Beyotime Institute of Biotechnology, China) with a phosphorylated protease inhibitor cocktail (Yeasen, Shanghai, China) and a proteinase inhibitor cocktail (Yeasen, Shanghai, China). Protein concentrations were determined using a BCA protein quantification kit (Yeasen, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were incubated with primary antibodies overnight at 4 °C and probed with secondary antibodies at room temperature for 1–2 h. The following antibodies were used: anti-KSR2 (1:500, Abnova, Taiwan, China), anti-CRAF (1:1000, CST, Massachusetts, USA), anti-p-CRAF (1:1000, CST, Massachusetts, USA), anti-MEK1/2 (1:1000, CST, Massachusetts, USA), anti-p-MEK1/2 (1:1000, CST, Massachusetts, USA), anti-ERK1/2 (1:1000, CST, Massachusetts, USA), anti-p-ERK1/2 (1:1000, CST, Massachusetts, USA), anti-14–3-3ζ (1:1000, CST, Massachusetts, USA), anti-Cleaved-Caspase 3 (1:500, Arigo, Taiwan, China), and anti-β-actin (1:1000, CST, Massachusetts, USA).
3
Molecular Cloning and Cell Culture Protocol
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High fidelity restriction endonucleases BamHI-HF, HindIII-HF, EcoRI-HF, XbaI-HF, and T4 ligase enzyme were purchased from NEB (New England Biolabs, MA, USA). Bacteria strain DH10B, BL21(DE3), Taq DNA polymerase, and All-in-One First-Strand cDNA Synthesis SuperMix for qPCR were purchased from Transgen, and JM109(DE3) was purchased from Promega (Promega, WI, USA). pT7Oi and pT7Om vectors were already constructed by our group. psilence-2.1-U6-Hygro vector was purchased from Beijing Rambolide Trading Co. DNA fragments were purified with AxyPrep DNA Gel Extraction Kit (Axygen) or DNA Clean-up Kit (Cwbio), and plasmids were extracted by FastPure Plasmid Mini Kit (Vazyme) or Endo-Free Plasmid Mini Kit I (Omega). Cell RNA Kit, BCA protein quantification kit, and Hieff Trans liposomal transfection reagent were purchased from Yeasen. ProtLytic Protein Lysis and Sample Loading was purchased from New Cell & Molecular biotech Co. Human embryonic kidney 293T (HEK293T) was a gift from Professor Yao Shaohua’s laboratory. Michigan Cancer Foundation-7 (MCF-7) and human hepatocellular carcinomas (Hep G2) were purchased from ATCC (American Type Culture Collection, VA, USA).
4
Western Blot Analysis of Cellular Proteins
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Cellular proteins were extracted with a mixture of RIPA lysis buffer (Solarbio, Beijing, China) and protease inhibitor (Solarbio, Beijing, China). Protein concentrations were determined using the BCA protein quantification kit (Yeasen, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with primary antibodies at 4°C overnight using the following primary antibodies: GAPDH (CST, USA), E-cadherin (CST, USA), N-cadherin (CST, USA), Vimentin (CST, USA), HIF-1α (CST, USA), VEGF-A (CST, USA) and incubated with horseradish peroxidase-linked secondary antibody (CST, USA) for 1 to 2 hours at room temperature, followed by 3 washes with TBST for 5 minutes. The signals were detected using the ECL chemiluminescence system and the grayscale values of each target band were analyzed by ImageJ software.
5
Protein Analysis by Western Blotting
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Proteins were extracted with Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, China), and their concentrations were checked with BCA Protein Quantification Kit (Yeasen, China). After being separated using SDS-PAGE with PAGE Gel Quick Preparation Kit (12.5% and 10%) (Yeasen, China) and transferred onto the PVDF membrane (GE, USA), the unreacted sites were blocked with 5% skim milk (Beyotime, China) for 2 h and the primary antibodies against SLC7A5 (1 : 200, goat, ab99419), β-actin (1 : 500, goat, ab8229; 1 : 1000, rabbit, ab8227), p-mTOR (phospho-S2448, 1 : 1000, rabbit, ab109268), mTOR (1 : 1000, rabbit, ab32028), p-S6K1 (phospho-S424, 1 : 500, rabbit, ab131436), S6K1 (1 : 5000, rabbit, ab32529), p-4EBP (1 : 500, rabbit, ab47365), and 4EBP (1 : 2000, rabbit, ab32024) (Abcam, USA) were added and the samples were incubated at 4°C for 12 h. After being rinsed three times with TBST (Yeasen, China), the secondary HRP-conjugated donkey anti-goat antibody (1 : 1000, ab6885) or goat anti-rabbit antibody (1 : 2000, ab6721) (Abcam, USA) was administrated to the membrane and maintained for 1 h at room temperature. Next, the bands were washed with TBS-T and visualized using the 4CN HRP kit (Leagene, China). The relative expression levels were obtained via ImageJ 1.53f (NIH, USA).
6
Immunoprecipitation and Western Blot Analysis
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Cells were scraped following treatment and the whole-cell protein was extracted with NP40 lysis buffer (P0013F, Beyotime, China) supplemented with protease inhibitors. Protein concentrations were measured using a BCA Protein Quantification Kit (20201ES76, YEASEN, China). The whole-cell protein was incubated with the relevant antibody or the control IgG (2729, CST, USA) overnight at 4 °C. The next day, protein A/G magnetic beads (HY-K0202-1, MCE) were added into the mixture for 2.5 h at 4 °C. Subsequently, the beads were washed five times with PBST (1×PBS + 0.5% Tween-20, pH 7.4) and were boiled. After that, the protein samples were separated electrophoretically and transferred to polyvinylidene fluoride membranes (ISEQ00010, Merck Millipore, USA). Then, 5% non-fat milk (A600669, Sangon Biotech, China) was used for blocking. The primary and secondary antibodies used in this study were listed in Table S3 .
7
Protein Extraction and Western Blot
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The total proteins were extracted from HEK 293T cells after 48 hr of culture. Protein concentration was determined using BCA Protein Quantification Kit (purchased from Yeasen Biotech Company Limited, Shanghai, China) under the guidance of the instruction. 10 μg of proteins were separated on 10% of SDS–PAGE and transferred electrophoretically to the nitrocellulose membrane. Proteins were probed by monoclonal anti‐GFP, anti‐UPF1, or anti‐β‐actin antibody overnight.
8
Molecular Mechanisms of DSS-Induced Colitis
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Dextran sodium sulfate (DSS) was purchased from MP Biomedicals (Irvine, CA, United States), and salazosulfapyridine (SASP) was purchased from Xinyi Pharmaceutical Group (Shanghai, China). BT2 was obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). The LEGENDplex™ Multi-Analyte Flow Assay Kit was supplied by BioLegend (San Diego, CA, United States). The BCA Protein Quantification Kit and Supper ECL Detection Reagent were purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China). The antibodies used for these experiments were as follows: Ribosomal protein S6 (S6) [1:1000, #2317; Cell Signaling Technology (CST), Danvers, MA, United States], phosphorylated S6Ser235/236 (p-S6, 1:1000, 4858; CST), eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) (9644, 1:1000; CST), p-4EBP1Thr37/46 (1:1000, 2855; CST), COX-2 (1:1000, 12282; CST), mTOR (1:1000, 66878-1-Ig; Proteintech, Rosemont, IL, United States), p-mTORSer2448 (1:1000, 67778-1-Ig; Proteintech), branched-chain amino transferase 2 (BCAT2) (1:1000, 16417-1-AP; Proteintech), rabbit antibody (1:10000, SA00001-2; Proteintech), mouse antibody (1:10000, SA00001-1; Proteintech), BCKDK (1:500, 374424; Santa Cruz, Dallas, TX, United States), and branched-chain α–keto acid dehydrogenase (BCKDHA) (1:500, 271538; Santa Cruz).
9
RARS1 Expression Validation via qPCR and Western Blot
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Cells were collected 48‐h posttransfection. The TRIzol method was used to extract total RNA. After DNA digestion, cDNA synthesis was performed by reverse transcription, and RARS1 expression was validated by qPCR. Total protein was extracted from cells using the RIPA lysis buffer, and protein concentration was measured using the BCA Protein Quantification Kit (Yeasen Biotechnology, SH, China). Proteins were denatured, and ArgRS expression was confirmed by Western blot using the HA‐Tag monoclonal antibody (Dia‐an, WH, China).
10
Quantifying Protein Expression via SDS-PAGE and Western Blot
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The protein samples were isolated from cells via a total protein extraction kit (Bohu, Shanghai, China). The BCA protein quantification kit (Yeason, Shanghai, China) was used to confirm the concentration of each protein specimen. Each channel of SDS/PAGE electrophoresis was added 50 µg of protein sample. The gel with target gene was transferred to the PVDF membrane and blocked with 5% skim milk. Then, the membrane was incubated with rabbit polyclonal anti-NOX 4 (1:2000) overnight followed by goat anti-rabbit for two hours. The specific proteins were visually detected using a chemiluminescence reagent. ImageJ software was applied to quantify protein expression.
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