Cultured cells were washed and lysed by Pierce RIPA buffer (Thermo Fisher Scientific, Madison, WI, USA). Protein lysates were homogenized and then underwent centrifugation. The supernatant was used for the analysis. The protein concentration was calculated using the Pierce BCA Protein Assay Kit (Takara Bio, Ohtsu, Japan). NuPAGE LDS sample buffer (Invitrogen) was added to each adjusted protein sample and resolved on 10% sodium dodecyl sulfate polyacrylamide gel. Electrotransfer was performed to polyvinylidene fluoride membranes using the iBlot Gel Transfer Device (Invitrogen) and blocked in 5% nonfat dry milk. Membranes were immunoblotted overnight at 4°C with a mouse anti-COL1A1 antibody (SAB1402151; Sigma–Aldrich, St. Louis, MO) followed by peroxidase-conjugated secondary antibodies. For β-actin, a mouse monoclonal anti-β-actin antibody (Abcam, Cambridge, UK) was used. Signals were detected by enhanced chemiluminescence (Lumivision PRO HSII, Aisin Seiki, Kariya, Japan).
Pierce bca protein assay kit
Manufactured by Takara Bio
Sourced in Japan
The Pierce BCA Protein Assay Kit is a colorimetric detection method used to quantify total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reaction to measure the reduction of copper ions by proteins in an alkaline environment, producing a purple-colored complex that absorbs light at 562 nm. The kit provides the necessary reagents and protocols to perform this assay.
Automatically generated - may contain errors
Lab products found in correlation
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti col1a1 antibody, by Merck Group (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Mettl13, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Mithras lb 940, by Berthold Technologies (1 mentions)
Atp bioluminescence assay kit cls 2, by Roche (1 mentions)
Easysee western blot kit, by Transgene (1 mentions)
5 protocols using pierce bca protein assay kit
1
Western Blot Analysis of Collagen I
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from cultured GC cells with RIPA buffer, and the supernatants were diluted in 5×SDS loading buffer after calculating protein concentration with a Pierce BCA protein assay kit (Takara, Japan). Western blot assay was carried out in accordance with the previous report (Yu et al. 2019 (link)). The signal bands were detected by Odyssey Infrared Imaging System (Li-COR Biosciences) based on manufacturer's instructions. The primary antibodies used in this study are as follows: METTL13 (#ab186002, Abcam), HN1L (#ab200571, Abcam), eEF1A (#2551, Cell Signaling Technology), β-actin (#81178, Santa Cruz Biotechnology).
3
ATP Bioluminescence Assay Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATP was measured by means of an ATP Bioluminescence Assay Kit CLS II (Roche Diagnostics, Tokyo, Japan), as per the manufacturer’s instructions. Luminescence was measured with a Mithras LB940 multimode microplate reader (Berthold, Tokyo, Japan). The total amount of ATP was normalized for total protein level using a Pierce BCA Protein Assay Kit (TaKaRa, Kyoto, Japan).
4
Quantifying Recombinant CTSL Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant CTSL protein was quantified using Pierce BCA Protein Assay Kit (Takara) and the 20–30 μg sample per lane was subjected to western blot analysis. Briefly, PVDF membranes were incubated for 2 h with mouse anti-Cathepsin L antibody [33/2] (1:1000 dilution in PBST) (Abcam, USA) and horseradish peroxidase (HRP)-conjugated goat anti mouse IgG (HRP) (1:2000 dilution in PBST) (Abcam, USA), detected by EasySee Western Blot Kit (TransGen Biotech, Beijing, China).
5
Exosome Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown to 80% confluent in complete medium and then in RPMI 1640 medium containing 2% exosome-depleted FBS for 48 h. The medium containing exosomes was collected and centrifuged for 10 min at 500×g, 20 min at 2000×g, and 30 min at 10,000×g. The exosomes pellet was harvested after 70 min of ultracentrifugation at 100,000×g (Beckman, Fullerton, CA) and then washed with PBS and ultracentrifugation again at 100,000×g for 70 min. Exosomes were then resuspended in PBS or RIPA for further studies. The concentration of exosomes suspension was measured by using Pierce™ BCA protein assay kit (Takara).
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!