B16F10 melanoma cells (5 × 104 cells/mL) were treated with the indicated concentrations of GABA (0–20 mM) in the presence or absence of 500 ng/mL α-MSH for 72 h. Total protein was extracted using a PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology), and total proteins were quantified using a Bio-Rad Protein Assay Kit (Bio-Rad). Western blotting was performed to detect the expression of MITF, tyrosinase, and pCREB according to our previous study [44 (link)]. β-Actin was used as an internal control.
Pro prep protein extraction solution kit
Manufactured by iNtRON Biotechnology
Sourced in Cameroon
The PRO-PREP Protein Extraction Solution Kit is a laboratory product designed to extract proteins from biological samples. It contains a proprietary solution that facilitates the isolation and purification of proteins from various sources, such as cells, tissues, or microorganisms. The kit provides a simple and efficient method for protein extraction, making it a useful tool for researchers and scientists working in the field of proteomics and biochemistry.
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Lab products found in correlation
Ecl prime western blotting detection system, by GE Healthcare (4 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Bis tris mini gel, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Pierce bicinchoninic acid bca protein assay, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Histone h1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Imagequant las 4000 instrument, by GE Healthcare (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
6 protocols using pro prep protein extraction solution kit
1
GABA Modulation of Melanoma Protein Expression
2
RNase H Activity Assay in A549 and HT-29 Cells
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The A549 and HT-29 tumor cell lines were supplied by the Korean Cell Line Bank (Seoul, South Korea) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Welgene, Gyeongsan, South Korea) supplemented by 10% fetal bovine serum (FBS) (RMBIO®, Missoula, MT, USA) in a humidified atmosphere with 5% CO2 at 37 °C. The cells were harvested during the exponential growth phase and diluted to make a 5 × 106 cells mL−1 solution. From the cell lysates, proteins were extracted based on the PRO-PREP™ protein extraction solution kit (Intron Biotechnology, Seongnam, South Korea), and the protein concentrations were calculated based on the Pierce™ bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Finally, RNase H activities included in 0.5 μg of each cell lysate were determined following the procedure shown in section ‘Procedure for RNase H activity assay’.
3
Western Blot Analysis of Protein Expression
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Transfected cells were incubated for 96 h. Total protein was extracted using the PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea) and nuclear protein was extracted with the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Kanagawa, Japan). The proteins were separated on 4–12% Bis-Tris Mini Gels (Life Technologies Corporation, Carlsbad, CA, USA) and transferred to membranes using an iBlot Dry Blotting System (Life Technologies Corporation). The membranes were incubated overnight at 4°C with mouse monoclonal antibodies against STMN1 (1:1000; Santa Cruz Biotechnology), p27 (1:1000; Santa Cruz Biotechnology), β-actin (1:1000; Sigma, St Louis, MO, USA) and Histone H1 (1:1000; Santa Cruz Biotechnology). The membranes were then treated with horseradish peroxidase-conjugated anti-mouse secondary antibodies and the proteins were detected with the ECL Prime Western Blotting Detection System (GE Healthcare, Tokyo, Japan).
4
KPNA2 Expression in Cholangiocarcinoma
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Western blot analysis was used to confirm KPNA2 protein expression in cholangiocarcinoma cell lines. Total proteins, nucleoproteins, and cytoplasmic proteins were extracted from TFK1 and HuCCT1 cells using the PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea) and the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). The proteins were separated using SDS–PAGE with 10% Bis–Tris gels and transferred to membranes. The membranes were blocked with 5% skim milk and incubated overnight at 4°C with anti-KPNA2 rabbit polyclonal antibody (1:1000; Abcam), anti-MRE11 rabbit monoclonal antibody (1:2000; Abcam), anti-RAD50 mouse monoclonal antibody (1:2000; Abcam), anti-p95 NBS1 rabbit monoclonal antibody (1:2000; Abcam), GAPDH antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-β-actin mouse monoclonal antibody (1:1000; clone AC-74; Sigma, St. Louis, MO, USA). The membranes were then treated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Specific signals were detected using the ECL Prime Western Blotting Detection System (GE Healthcare, Tokyo, Japan) and quantified using an Image Quant LAS 4000 instrument (GE Healthcare).
5
Quantifying HSP110 and β-Actin Expression
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Western blotting was used to evaluate HSP110 and β-actin expression in MKN45 cells. MKN45 cells were treated with KNK437 at 43 °C for 3 h. Then, total proteins were extracted using the PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology, Sungnam, Kyungki-Do, South Korea). Proteins were separated on a 10% polyacrylamide gel and transferred to nitrocellulose membranes using a wet transfer protocol. The membranes were incubated overnight at 4 °C with rabbit monoclonal antibody against HSP110 (1:1000; GeneTex) and β-actin (1:1000; Sigma, St Louis, MO, United States). The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies, and each proteins were evaluated using the ECL Prime Western Blotting Detection System (GE Healthcare, Tokyo, Japan) using Image Quant LAS4000 (GE Healthcare Life Sciences, United Kingdom).
6
HSP110 Expression Profiling in Gastric Cancer
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Western blotting was performed to confirm the expression of HSP110 and β-actin in gastric cancer cell lines. Transfected cells were incubated for 72 h, and total proteins were extracted from MKN7, MKN45, MKN74, and AZ521 cells using PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea). The proteins were separated on 4–12% Bis-Tris Mini Gels (Life Technologies Corporation, Carlsbad, CA, USA), and transferred to membranes using an iBlot Dry Blotting System (Life Technologies Corporation, Carlsbad, CA, USA). The membranes were incubated overnight at 4°C with rabbit monoclonal anti-HSP110 antibody (1:1000; GeneTex, CA, USA) and anti-β-actin antibody (1:1000; Sigma-Aldrich, St Louis, MO, USA). Following this, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies, and the target proteins were detected with the ECL Prime Western Blotting Detection System (GE Healthcare, Tokyo, Japan) using Image Quant LAS4000 (GE Healthcare Life Sciences, UK).
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