Cells were suspended and homogenized with ten times volume of Homogenize solution (HS; 20 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 100 μg/mL DNase, 50 μg/mL RNaseA, 1 mM PMSF, and protease inhibitor cocktail). Homogenized samples were pre-incubated with Protein A-Sepharose 4B beads (Sigma-Aldrich) that were previously incubated with NOX2 antibody or normal rabbit IgG. An equal volume of sample (1 mg) and NOX2 or normal rabbit IgG-bound beads were incubated at 4 °C for 4 h. After the incubation, beads were washed three times with HS containing 1 mg/mL BSA. The washed beads were mixed with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 6% 2-mercaptoethanol, and 20% glycerol) to extract NOX2-bound proteins. Extracted samples were analyzed by SDS-PAGE and western blotting as described above.
Protein a sepharose 4b beads
Manufactured by Merck Group
Protein A-Sepharose 4B beads are a chromatography resin composed of protein A covalently coupled to Sepharose 4B beads. Protein A is a bacterial protein that has a high affinity for the Fc region of immunoglobulins, making the beads useful for the purification of antibodies and antibody-containing samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Bradford analysis, by Bio-Rad (2 mentions)
Supersignal chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Ne pertm nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti gamma h2a x phospho s139 antibody, by Abcam (1 mentions)
Parp 1 antibody f 2, by Santa Cruz Biotechnology (1 mentions)
6 protocols using protein a sepharose 4b beads
1
Immunoprecipitation and Western Blot Analysis of NOX2 Protein
2
Nucleoprotein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of the extracted cells was lysed in frozen cell lysis buffer (NEB) containing 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma). The lysate was pre-cleared with 15×l protein A Sepharose 4B beads (Sigma) at 4° C for 30 minutes. NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, 78833) were used to extract the nucleoprotein of the cell. The pre-determined BCA (Pierce, Rockford, IL) and Western Blot procedures were as described in the literature. (Vinculin Antibody, Rabbit Polyclonal, Affinity Biosciences Cat# AF5122, RRID:AB_2837608, EPCAM Antibody, Rabbit Polyclonal, Affinity Biosciences Cat# 21050-1-AP)
3
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cell lysates were prepared using a 9M Urea, 0.075 M Tris buffer (pH 7.6), and the Bradford Assay was used to quantify protein lysates. For Western blot anaylsis, 50–100µg of protein as determined by Bradford analysis (BioRad), was subjected to reducing SDS/PAGE using standard methods. For immunoprecipitation, five hundred micrograms of whole-cell lysate was incubated with DDR2 antibody, and captured using protein A-sepharose 4B beads (Sigma). Blots were probed with indicated antibodies, followed by corresponding horseradish peroxidase-conjugated secondary antibodies. Detection was preformed using SuperSignal Chemiluminescent substrate (Thermo Scientific) on a ChemiDoc XRS+(BioRad). Integrated relative densities of individual bands were quantified using ImageLab software. All quantification was prefomed under conditions of linear signal response.
4
Protein Extraction and Analysis from Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of the extracted cells was lysed in ice-cold cell lysis buffer (NEB) containing 1 mM PMSF and 1:100 protease inhibitor cocktail (Sigma). The lysate was pre-cleared with 15 × l protein A Sepharose 4B beads (Sigma) at 4°C for 30 min. NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833) were used to extract nucleoprotein from cells. The BCA protein assy (Pierce, Rockford, IL, United States) and Western Blot procedures were performed as described previously (Ruiz et al., 2019 (link)). Antibodies used included Anti-beta Actin antibody (1:50000, Abcam, ab49900), Anti-gamma H2A.X (phospho S139) antibody (1:1000, Abcam, ab2893), PARP-1 antibody (F-2) (1: 500, Santa Cruz, sc-8007), Cdc2 p34 antibody (17) (1:500, Santa Cruz, sc-54), BRCA1 antibody (D-9) (1:500, Santa Cruz, sc-6954), Phospho-cdc2 (Tyr15) Antibody (1:1000, Cellsignal, #9111), Phospho-BRCA1 (Ser1497) Polyclonal Antibody (1:1000, Thermo Fisher Scientific, # PA5-64621), Rad51 Antibody (G-5) (1:500, Santa Cruz, sc-133089), XRCC1 (1:1000, Abcam, ab44830), and Lamin B1 (1: 20000, Proteintech, 66095-1-Ig).
5
Immunoprecipitation of V5-tagged Rad51 and Rad52
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
V5-tagged versions of Rad51 and Rad52 were immunoprecipitated using 10 µl of anti-V5 and 100 µl of Protein A-Sepharose 4B beads (Sigma). Beads were incubated with antibodies for 1 h, added to 0.8 ml of crude extracts (around 3 mg of total protein) and incubated overnight at 4°C. Beads were washed four times with TBS-T. Proteins were eluted by resuspension of beads in 10 µl of 2x SDS gel loading buffer and incubation at 95°C for 5 min for analysis by western blot.
6
Quantitative Western Blot Analysis
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!