Total protein of cell lines was isolated using a total protein extraction kit (Boster Biological Technology, Wuhan, China); concentration of protein was determined using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.) at 570 nm absorption value. Western blotting was conducted as described by Li et al. [9 (link)]. Briefly, lysate protein was separated using SDS-PAGE, using 10% gel, and was transferred to nitrocellulose membrane. The expression signal was detected using an enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA). Primary antibodies used in this assay were as follow: iASPP primary antibody (ab115605, Abcam, UK), GAPDH primary antibody (ab9385, Abcam, UK), and anti-rabbit secondary antibody (ab191866, Abcam, UK).
Total protein extraction kit
Manufactured by Boster Bio
Sourced in China
The Total Protein Extraction Kit is a tool designed for the efficient extraction and purification of total protein from various biological samples. The kit provides a standardized protocol and necessary reagents to facilitate the extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence system, by Merck Group (1 mentions)
Ab115605, by Abcam (1 mentions)
Ab9385, by Abcam (1 mentions)
Ab191866, by Abcam (1 mentions)
Gs 710, by Bio-Rad (1 mentions)
Nf κb p65 primary antibody, by Santa Cruz Biotechnology (1 mentions)
Bicinchoninic acid protein assay kit, by Boster Bio (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Chloral hydrate, by Sangon (1 mentions)
Unlq 10 column total rna isolation kit, by Sangon (1 mentions)
Malondialdehyde mda assay kit tba method, by Nanjing Jiancheng (1 mentions)
Total nitric oxide assay kit, by Beyotime (1 mentions)
Alanine aminotransferase alt assay kit, by Nanjing Jiancheng (1 mentions)
Aspartate aminotransferase ast assay kit, by Nanjing Jiancheng (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
High sig ecl western blotting substrate, by Shanghai Tanon Science & Technology (1 mentions)
Con a, by Solarbio (1 mentions)
5 protocols using total protein extraction kit
1
Protein Isolation and Western Blotting
2
Western Blot Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted using the Total Protein Extraction Kit (Boster, Wuhan, China) and phenylmethylsulfonyl fluoride (100:1) (Solarboi, Beijing, China). The BCA assay was used to measure protein concentrations in order to normalize sample loading for separation on 10% SDS-PAGE. Separated proteins were transferred onto PVDF membranes and blocked with 5% BSA for 1 h. It was then incubated overnight at 4°C overnight with primary antibodies, washed with TBST for 3 × 15 min, and incubated for 1 h at 20°C with secondary antibodies. Protein bands were detected using enhanced chemiluminescence (ECL). GAPDH was used as the loading control. The antibodies used in this study are listed in Table 3 .
3
Quantifying NF-κB p65 Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were purchased from pancreatic tissue using a total protein extraction kit (Boster, Wuhan, China). Protein concentrations were determined using a commercial kit (Pierce, Rockford, IL, USA). Each 20-μg aliquot of total protein was loaded onto sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred onto membranes. Following complete protein transfer, the membranes were blocked with 5% milk powder solution for 2 h and incubated with NF-κB p65 primary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight. After washing the membranes, the secondary antibody (Boster, Wuhan, China) was applied and incubated for 2 h at room temperature (RT). Bands were quantified by a calibrated imaging densitometer (GS-710; Bio-Rad) and analyzed by “Quantity One” software (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.
4
CTD Dose-Dependent Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MNNG/HOS and MG-63 cells were each treated with different concentrations of CTD (0–4 mg/ml) and (0–6 mg/ml) at the exponential growth phase for 24 h. Then, the total protein was extracted using a total protein extraction kit (Boster Biological Technology, USA). The protein level was quantified with a bicinchoninic acid protein assay kit from Boster according to the manufacturer’s protocol. The protein extract was separated using a 10–15% SDS–polyacrylamide gel electrophoresis and then transferred to the polyvinylidene fluoride (PVDF) membranes, then membranes were blocked in 5% skimmed milk dissolved in TBST for 1 h. The membranes were incubated at 4°C overnight with a primary antibody containing GAPDH, Akt, Bcl-2, Bax, PARP, and phosphor-Akt (Thr308), p-Cdc2, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibody at room temperature for 1 h. The protein expression was determined using enhanced chemiluminescence reagents (Millipore, Waltham, MA, USA). GAPDH was used as an internal control.
5
Oxidative Stress Evaluation in Animal Models
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Con A was bought from Solarbio Science & Technology Co., Ltd. (Beijing, China, catalog number: C8110). Malondialdehyde (MDA) assay kit (TBA method), Alanine aminotransferase (ALT) Assay Kit, and Aspartate aminotransferase (AST) Assay Kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China, catalog number: A003-1-2, C009-2-1 and C010-2-1). Total Nitric Oxide (NO) Assay Kit was gotten from Beyotime Biotechnology (Shanghai, China, catalog number: S0021S). Chloral hydrate, UNlQ-10 Column Total RNA Isolation Kit and 2X SYBR Abstart Master Mix was purchased from Sangon Biotech Co., Ltd. (Shanghai, China, catalog number: A600288, B511321 and B110032). The Total Protein Extraction Kit (BOSTER Biological & Technology Co. Ltd., China, catalog number: AR0146), SDS–polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime Biological Technology Co. Ltd., China, catalog number: P0012A), PVDF membranes (Millipore, USA, catalog number: R1CB73920), High-sig ECL Western Blotting Substrate (Tanon Science & Technology Co., Ltd., China, catalog number:180-501), Anti-p38 Rabbit pab, Mapk3k13 Rabbit pab, HRP-conjugated Goat Anti-Rabbit IgG (Additional file 1 : Table S1) were used to detect the expression of target protein.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!