RIPA buffer (KeyGen Biotech. Co., Ltd.) was used to collect total protein. Protein concentration was determined by BCA reagent (KeyGen Biotech). Spacer gel (5%) and 10% separation gel were prepared using an SDS-PAGE gel kit (Beijing, Solarbio Science & Technology Co., Ltd.). An equal amount of each protein (30 µg) was loaded to the gel and electrophoresis was performed on a Bio-Rad Mini-PROTEAN system (Bio-Rad Laboratories, Inc.). Proteins were transferred to 0.45 µm PVDF membranes (EMD Millipore) using a semi-dry transfer system (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% BSA at room temperature for 1 h and incubated with the following rabbit monoclonal primary antibodies (Cell Signaling Technology, Inc.) at 4°C overnight: PTEN (1:1,000; product no. 9188), Akt (1:1,000; product no. 4691), p-Akt (1:1,000; Ser473; product no. 4060) and β-actin (1:3,000; product no. 4970). After membranes were incubated with an HRP-conjugated secondary antibody (1:3,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.) for 45 min at room temperature, blotting signals were visualized with ECL substrates (EMD Millipore). Densitometric analysis was conducted using Quantity One software (version 4.6; Bio-Rad Laboratories) and protein expression was normalized to endogenous β-actin.
Bca reagent
Manufactured by Keygen Biotech
Sourced in China
The BCA reagent is a colorimetric detection kit used for the quantitative determination of total protein concentration. It is a common laboratory tool utilized in various biochemical assays and studies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ecl reagent, by Advansta (2 mentions)
Ab197297, by Abcam (2 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Ripa buffer, by Keygen Biotech (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Biorad miniprotean system, by Bio-Rad (1 mentions)
Rabbit monoclonal primary antibodies, by Cell Signaling Technology (1 mentions)
Sds page gel kit, by Solarbio (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Sds page loading buffer, by Biosharp (1 mentions)
Ab168352, by Abcam (1 mentions)
4 protocols using bca reagent
1
Western Blot Analysis of PTEN/Akt Pathway
2
Western Blot Analysis of Protein Expression
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The pre-treated NUGC-4 cells were scraped and lysed with radioimmunoprecipitation assay (RIPA) buffer containing 1:100 completeTM Protease Inhibitor Cocktail (Roche, United States) in 1.5 ml EP tubes on ice for 20 min. Then, the lysed samples were centrifuged at 12,000 rpm for 20 min at 4°C. After centrifugation, the supernatants were transferred to clean EP tubes and mixed with 1:4 volume 5 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (BioSharp Inc., Nanjing). The samples were placed in boiling water for 5 min to ensure protein denaturation. Protein was quantified using bicinchoninic acid (BCA) reagent (Keygen). Subsequently, equal amounts of proteins (15 μg) from each sample were added to SDS-PAGE to separate the target protein, and then transferred onto polyvinylidene fluoride (PVDF) blotting membranes (GE Healthcare, Germany). The polyacrylamide gels were prepared in advance using a PAGE Gel Fast Preparation Kit (Epizyme Inc., Nanjing). Next, the membranes were blocked with 5–10% skim milk for 1 h, then cut into strips and incubated with the primary antibodies overnight and subsequently with the secondary antibodies for 1 h. Protein bands were visualized according to the instructions of the PageRuler pre-stained protein ladder (Thermo Fisher Scientific, United States).
3
Western Blot Analysis of Protein Expression
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Cells were lysed by using RIPA buffer supplemented with 1% PMSF for preparing whole-cell extracts. Protein concentration was quantified with BCA reagent (KeyGen Biotech, Nanjing, China) and equal amounts of total proteins were separated on 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Then, the membranes were blotted with antibody against MCCC2 (ab197297, Abcam, 1:500), GLUD1 (ab168352, Abcam, 1:500), GLUD2 (39,038, SAB, 1:500) or p38 MAPK (9212, CST, 1:500). GAPDH (2118, CST, 1:1000) was used to normalize the protein level. Visualization was then carried out using their own secondary antibodies (Affinity Biosciences. OH. USA) and ECL reagents (Advansta, USA).
4
Western Blot Quantitation of CDK3
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Whole-cell extracts were prepared by lysing the cells in RIPA buffer supplemented with 1% PMSF. The BCA reagent (KeyGen Biotech, China) was applied in quantitation of the protein concentration, then 10% SDS–PAGE was used to separate equal amounts of total proteins. Then, PVDF membranes (Millipore, USA) were used to transfer the proteins to membranes. We blotted the membranes with an CDK3 antibody (ab197297, Abcam, 1:500), and the protein levels were normalized with GAPDH (2118, CST, 1:1000). Second antibodies (Affinity Biosciences, USA) and ECL reagents (Advansta, USA) were then used to visualize.
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