Cells were lysed by using modified RIPA buffer containing 10 mM Tris-HCl at pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.025% SDS and proteinase inhibitors on ice for 10–20 min. Protein samples were quantified using BCA protein assay kit (Macgene, MPK002), boiled in 5 × loading buffer and resolved by SDS-PAGE and transferred onto nitrocellulose membrane. In all, 5% skimmed milk (BD Difco, 232100) in TBS supplemented with 0.1% Tween (TBST) was used to block the membrane before probing with indicated antibodies in TBST at 4 °C overnight. Membranes were washed with TBST and then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature for 1 h and developed with ECL Western Blotting Detection Reagent (ThermoFisher Scientific, 32132). Blot bands were quantified using ImageJ software. Uncropped scans of all the blots in this manuscript are shown in the Supplementary Information.
Bca protein assay kit
Manufactured by Macgene
Sourced in China
The BCA protein assay kit is a laboratory tool used for the quantitative determination of total protein concentration in a sample. It is based on the bicinchoninic acid (BCA) method, which relies on the reduction of copper ions by proteins in an alkaline environment, producing a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Total exosome isolation kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by ZSGB-BIO (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Ab32147, by Abcam (1 mentions)
A5441, by Merck Group (1 mentions)
5 protocols using bca protein assay kit
1
Western Blot Protein Extraction Protocol
2
Isolation and Characterization of Exosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 48 h posttransfection, a Total Exosome Isolation kit (Invitrogen, USA) was applied to isolate the total exosomes from the supernatant of ADSC culture medium and culture medium transfected with pcDNA-lnc-KCNQ1OT1 or NC (ADSCs-Exos, LV-KCNQ1OT1-Exos, or LV-NC-Exos, respectively) according to the manufacturer's protocol [26 (link)]. Bicinchoninic acid (BCA) protein assay kit (MACGENE, China) was used to measure the concentration of isolated exosomes. Before use, all exosome samples were analyzed for proper size by nanoparticle tracking analysis (NTA; NanoSight, Malvern) and for morphology by transmission electron microscopy (TEM). The protein levels of CD9, CD63, CD81, and Alix (representative markers of exosomes) were then detected.
3
Western Blot Analysis of p-AMPK in ApoE-/- Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins in apoE–/– liver were lysed in RIPA buffer containing a cocktail of protease and phosphatase inhibitors (Roche). After quantified with BCA protein assay kit (MACGENE, China), proteins were electrophoresed by SDS-PAGE on 10% gels and transferred to PVDF membrane (Millipore). The 5% BSA was used to block PVDF membranes. Then membranes were incubated with antibodies antibodies aganist p-AMPK, AMPK, β-actin (all from Cell Signaling Technology). Membranes were subsequently incubated with HRP-conjugated goat anti-rabbit secondary antibodies (ZSGB-BIO, China) and SuperSignal West Pico chemiluminescent substrate (Millipore).
4
Western Blotting of Protein Markers in Tissues and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed on paraffin sections as described in our previous study (27 (link)). Briefly, tissues and cells were lysed using RIPA buffer, and protein samples were quantified using a BCA protein assay kit (Macgene, MPK002) and boiled in 5× loading buffer. Then, total protein extracts from tissues and cells were analyzed using western blotting with antibodies against FAM72A (Proteintech, anti-rabbit, 21203-1-AP), mTOR (Cell Signaling Technology, anti-rabbit, mAb 2983), p-mTOR (1:1000, 5536, CST), PCNA (1:2000, 13110, CST), CCNE1/cyclin E1 (Abcam, ab71535), CCND1/cyclin D1 (Cell Signaling Technology, 55506), and CDK2 (Abcam, ab32147). An anti-β-actin antibody (Sigma, anti-rabbit, A5441) was used as the internal reference. Western blot results were quantified using ImageJ software.
5
Protein Expression Quantification by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression of proteins including γ-H2AX, Caspase-3, Cyclin B1, CDC-2, Phospho-CDC-2, CDK-4 and Cyclin D1 was quantified by use of western blot. Briefly, cells were seeded in 6-well plates for exposure to TDCPP for 24 hr, after which cells were harvested and lysed in the lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Macgene). Samples were centrifuged at 16900 g for 10 min at 4°C. Concentrations of protein were determined using the BCA Protein Assay Kit (Macgene). Each sample (30-40 μg) was loaded and electrophoresed on 10% SDS-PAGE. Proteins were transferred to PVDF mem-brane after electroblotting at 4°C. Subsequently, membranes were blocked with 5% skim milk and incubated with specific antibodies at 1:1000 dilution at 4°C overnight. Subsequently, membranes were then incubated with the HRP-conjugated goat anti-rabbit IgG or goat antimouse IgG secondary antibodies (1:5000) at room temperature for 1 hr. The protein signals were visualized by enhanced chemiluminescence (ECL) system and the total gray values of each band were analyzed using AlphaEase-FC software V3.1.2. Relative expression of each protein was calculated by normalization to β-actin, and the resulting ratios in the control group were normalized to 1.
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!