Purified PDCoV particles, mock-infected, or PDCoV-infected LLC-PK1 cell were lysed in lysis buffer (25 mM Tris-HCl, 200 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 25 mM β-glycerophosphate, 1% NP40) containing a protease inhibitor cocktail (Bimake, USA) and incubated on a shaker for 30 min at 4 °C. The lysates were then cleared by centrifugation at 14,000×g for 10 min. The supernatants were precipitated with anti-NS6-pt, rabbit IgG and anti-N mAb, respectively, and incubated with gentle rocking overnight at 4 °C. Protein A beads (Beyotime, China) washed with lysis buffer were added to supernatant fractions and incubated with gentle rocking for 2 h at 4 °C. After washing four times with lysis buffer, isolated immunoprecipitated proteins were boiled 10 min with PAGE sample loading buffer and then subjected to WB analysis with specific antibodies.
Protein a beads
Manufactured by Beyotime
Sourced in China
Protein A beads are a type of chromatography resin used for the purification of antibodies. They are composed of agarose beads with covalently coupled recombinant Protein A, which binds to the Fc region of immunoglobulins. Protein A beads can be used to capture and purify antibodies from complex mixtures, such as cell culture supernatants or serum samples.
Automatically generated - may contain errors
Lab products found in correlation
Cell lysis buffer, by Beyotime (5 mentions)
3xsds sample buffer, by Cell Signaling Technology (3 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Sds sample buffer, by Cell Signaling Technology (1 mentions)
Anti sod2, by Abcam (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
7 protocols using protein a beads
1
Immunoprecipitation and Western Blot Analysis of PDCoV Proteins
2
Immunoprecipitation from Liver Tissue Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The liver tissues were lysed with cell lysis buffer (Beyotime Company, P0013). Lysates were clarified by centrifugation at 12,000 g for 15 min and were used for immunoprecipitation. A total of 2 μg of antibody was incubated with 500-1000 μg of protein overnight at 4°C. Next, protein A beads (Beyotime Company, P2006) were added and the mixture was incubated overnight at 4°C. After incubation, the beads were washed 3 times, solubilized in 40 μl 3xSDS sample buffer (Cell Signaling Technology, 7722), and analyzed by western blotting.
3
Protein Immunoprecipitation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation experiments, cells lysate was incubated with 200 μL protein A beads (Beyotime) supplemented with primary antibody at 4°C overnight. After washing the protein A beads with 0.5 μL cold lysis buffer, normalized amounts of total cell lysates or immunoprecipitated samples were analyzed by western blot.
4
Cd-induced Protein Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells were treated with Cd and lysed with cell lysis buffer (Beyotime Company, P0013). Lysates were clarified by centrifugation at 12,000 g for 15 min and were used for immunoprecipitation. A total of 2 μg of antibody was incubated with 500–1000 μg of protein overnight at 4°C. Next, protein A beads (Beyotime Company, P2006) were added and the mixture was incubated overnight at 4°C. After incubation, the beads were washed 3 times, solubilized in 40 μl 3xSDS sample buffer (Cell Signaling Technology, 7722), and analyzed by western blotting.
5
Immunoprecipitation of proteins in HepG2 cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells were treated with PA in the presence or absence of RSV. Thereafter, cells were lysed with cell lysis buffer (P0013; Beyotime Institute of Biotechnology, Shanghai, China). Lysates were clarified by centrifugation at 12, 000 × g for 15 min and used for immunoprecipitation. A total of 2 mg of antibodies was incubated with 500-1000 mg of protein overnight at 4 °C. Next, protein A beads (P2006; Beyotime Institute of Biotechnology) were added and the mixture was incubated overnight at 4 °C. After incubation, the beads were washed three times, solubilized in 40 mL 3× SDS sample buffer (7722; Cell Signaling Technology), and analyzed by western blotting.
6
Evaluating TiCl4 Effects on Osteoblastic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteoblastic cells were treated with TiCl4 at different concentrations (0, 10, 25 and 50 µM) at 37°C for 24 h and lysed with cell lysis buffer (Beyotime Institute of Biotechnology; cat. no. P0013). Lysates were clarified by centrifugation at 12,000 × g for 15 min at 4°C and were used for immunoprecipitation. The protein concentrations were determined using a Bradford protein assay kit (Beyotime Institute of Biotechnology). A total of 2 µg anti-SOD2 (1:200; Abcam; cat. no. Ab137037) was incubated with 500-1,000 µg protein overnight at 4°C. This was followed by incubation with 20 µl protein A beads (Beyotime Institute of Biotechnology; cat. no. P2006) overnight at 4°C. After incubation, the beads were washed three times with PBS and subsequently boiled in 40 µl 1X sample buffer for 5 min at 100°C, and analyzed by western blotting.
7
Immunoprecipitation of Proteins from 5-ALA-Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with 5-ALA and photodynamic therapy, then lysed with cell lysis buffer (Beyotime Company, P0013). Lysates were clarified by centrifugation at 12,000 g for 10 min and were used for immunoprecipitation. A total of 2 mg of antibody was incubated with 500–1000 mg of protein overnight at 4 ℃. Next, protein A beads (Beyotime Company, P2006) were added and the mixture was incubated overnight at 4 ℃. The beads were washed 3 times, then solubilized in 40 ml 3xSDS sample buffer (Cell Signaling Technology, 7722), and analyzed by western blotting.
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!