The protein extracted by radio-immunoprecipitation assay lysis buffer (containing protease inhibitor) was quantified by bicinchoninic acid kit (Takara), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane (Bio-Rad, CA, USA). The blot was blocked in BSA and immunostained with primary antibodies HSP70 (1:1000), CD9 (1:1000), SEPT2 (1:1000Abcam, MA, USA), CD81(1:500; Santa Cruz Biotechnology, CA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000; CST, MA, USA) and a secondary antibody . Signals were detected by chemiluminescence [16 (link),30 (link)].
Bicinchoninic acid kit
Manufactured by Takara Bio
Sourced in United States, China
The Bicinchoninic acid (BCA) kit is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. The kit utilizes the reduction of copper ions by the protein in an alkaline medium and the subsequent colorimetric detection of the cuprous cations using the reagent bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Hsp70, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Ab155933, by Abcam (1 mentions)
Ab195049, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ab179467, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Ab32101, by Abcam (1 mentions)
Ab39636, by Abcam (1 mentions)
Polyvinylidene membrane, by Merck Group (1 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Anti pten, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti magi1, by Abcam (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
4 protocols using bicinchoninic acid kit
1
Extracellular Vesicle Protein Analysis
2
Western Blot Analysis of Signaling Pathways
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The PTC tissues and cells lysates were collected using RIPA buffer (Invitrogen, shanghai, China) and protease inhibitor cocktail (#5871; Beverly, MA, USA). Protein concentrations were calculated using Bicinchoninic Acid Kit (Takara; Merck KGaA) referring to the manufacturer’s protocols. Immunoblotting procedures followed the previously published method [13 ]. Equal amounts (60 µg) of total tissues or cellular proteins were added into loading buffer, boiled for 5 min, and separated on 10 % SDS-PAGE electrophoresis. Subsequently, the proteins were metastasized to nitrocellulose (NC) membranes. Then we blocked the membranes with 5 % non-fat dry milk for 1 h at room temperature and hatched overnight with primary antibodies. Following further incubated with secondary antibody for 1 h, the immunoreactive protein strips were visualized by ECL kit (Thermo, CA, USA) on the Bio-Rad Chemical Doc XRS system (Bio-Rad). The primary antibodies were including anti-p-p38 (ab195049; 1:2000), anti-p38 (ab250612; 1:1000), anti‐p-JAK2 (ab32101; 1:1500), anti‐JAK2 (ab39636; 1:800), anti‐p-STAT1 (ab215820; 1:1000), anti‐STAT1 (ab155933; 1:1000) and anti‐β‐actin (ab179467; 1:5000) were purchased from Abcam.
3
Protein Expression Analysis in Glioma
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Glioma tissues or cells were homogenized and lysed with radioimmunoprecipitation assay lysis buffer supplemented with 1 mmol/l phenylmethylsulfonyl fluoride and phosphatase inhibitor (Pierce; Thermo Fisher Scientific, Inc, IL), and then centrifuged at 12,000 rpm for 10 min at 4°C. Protein concentration was measured using a bicinchoninic acid kit (Takara Biotechnology, Dalian, China). Immunoblotting was performed according to the standard protocol and the proteins were transferred to polyvinylidene membranes (Millipore, Billerica, MA, USA), blocked with TBST with 5% skimmed milk for 2 h at room temperature, and then incubated at 4°C overnight with either anti-MAGI1, anti-E-cadherin, anti-N-cadherin, anti-Bax, anti-Bcl2, anti-caspase3 (all 1:1,000, Abcam, Cambridge, MA, UK), anti-vimentin, anti-PTEN, anti phospho-Akt, anti β-catenin, anti-cyclin D1 (all 1:1,000, Cell Signaling Technology, Beverly MA, USA), or anti-GAPDH antibodies (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three washes in TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated IgG antibody for 2 h at room temperature. Immunoblot bands were quantified relative to GAPDH using Image Pro Plus software (Media Cybernetics, Inc.).
4
Protein Expression Analysis by Western Blot
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Total protein, which were extracted from tissue or cells, were quantitated for protein concentration using a bicinchoninic acid kit (Takara), followed by being electro-resolved in 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were then incubated with primary antibodies overnight at 4°C. The following antibodies used were from Abcam (Cambridge, MA, U.S.A.): HOXD9 (ab90260), E-cadherin (ab40772), N-cadherin (ab18203), Vimentin (ab8978), and GAPDH (ab8245), using dilutions recommended by the supplier. Following this, the membrane was incubated with the corresponding HRP linked anti-rabbit IgG antibody (1:2000, Cell signaling, Danvers, MA, U.S.A.) at room temperature for 1 h. The protein bands were visualized using the ECL-Plus reagent (Millipore, Billerica, MA, U.S.A.).
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