Centrifuge the positive cell lysates at 17,500 × g for 1 h at 4 °C.
{"action": "centrifuge", "output": "", "speed": ["17,500 \u00d7 g"], "time": ["1 h"], "temperature": ["4 \u00b0C"]}
Discard the pellets. #####%%%%% output "the pellets" are used in line6
{"action": "remove", "output": "the pellets", "reagent": [""]}
Divide the supernatants (soluble fractions) from step 1 into 10 µL aliquots at -80 °C. #####%%%%% output "the supernatants (soluble fractions)" are used in line10
{"action": "divide", "output": "the supernatants (soluble fractions)", "volume": ["10 µL"], "temperature": ["-80 °C"]}
Perform SDS-PAGE with the positive cell lysate to ensure the target protein is in the soluble fraction. #####%%%%% output "the BCA protein assay kit" are used in line10
{"action": "perform", "output": "the BCA protein assay kit", "device": ["SDS-PAGE"]}
Measure the total protein concentration in both positive cell lysates using the BCA protein assay kit according to the manufacturer’s instructions.
{"action": "measure", "output": "", "concentration": ["both positive cell lysates"], "reagent": ["the BCA protein assay kit", "the supernatants (soluble fractions)"]}
Rinse each new capillary with approximately four capillary volumes of 100 mM HCl, . #####%%%%% output "all future DNA library" are used in line16
{"action": "rinse", "output": "all future DNA library", "reagent": ["100 mM HCl"]}
Prepare 10 µL of 500 µM naive DNA library in SB.
{"action": "prepare", "output": "", "volume": ["10 µL"], "reagent": ["naive DNA library"]}
Use this treatment for all future DNA library .
{"action": "use", "output": "", "reagent": ["this treatment", "all future DNA library"]}
Prepare a 100 µM solution of the temperature-treated DNA library (from step 6) in 100 µL SB.
{"action": "prepare", "output": "", "concentration": ["100 µM"], "reagent": ["temperature-treated DNA library (from step 6)"]}
Prepare a 10 µL equilibrium mixture with 10 µM DNA library . #####%%%%% output "DNA" are used in line22
{"action": "prepare", "output": "DNA", "concentration": ["10 \u00b5M DNA library"]}
100 µM each masking DNA
{"action": "amplify", "output": "", "reagent": ["DNA"]}
Incubate for 10 minutes at room temperature. #####%%%%% output "the DNA library" are used in line26
{"action": "incubate", "output": "the DNA library", "temperature": ["room temperature"], "time": ["10 minutes"]}
Determine the migration time of the DNA library to the end of the capillary (free DNA component). #####%%%%% output "the free DNA" are used in line30
{"action": "determine", "output": "the free DNA", "reagent": ["the DNA library"]}
Perform NECEEM with the same equilibrium mixture.
{"action": "perform", "output": ""}
Determine the migration time of fluorescein to the end of the capillary to the free DNA.
{"action": "determine", "output": "", "reagent": ["fluorescein", "the free DNA"]}