A method for determining the relative content of protein or polypeptide
A relative content and protein technology, applied in the field of protein or peptide quantitative analysis, can solve the problems of high cost, long test time, difficulty in adapting internal reference protein or peptide to all samples, and small detection throughput, so as to achieve small investment and rapid method establishment Simple, simple and easy-to-operate experimental procedure
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Embodiment 1
[0031] 1.1 Preparation of peptide samples
[0032] The experimental samples were subjected to protein brandford quantification or cell calculation quality control. Take an equal or equal volume of samples and add them to peptide extraction lysate (8M urea / 100mM TEAB, pH 8.0, 1mM PMSF / 2mM EDTA), mix and incubate on ice for 5min, add DTT with a final concentration of 10mM was sonicated in an ice bath for 15min, then centrifuged at 13000g at 4°C for 30min, and the supernatant was transferred to a new centrifuge tube; DTT was added to the centrifuge tube to a final concentration of 10mM, and the reduction reaction was carried out in a water bath at 56°C for 30min. Get a reduced sample. Subsequently, IAM was added to a final concentration of 55 mM, placed in the dark at room temperature for 30 min for alkylation reaction, and the initial sample was obtained, treated with a 10 kD ultrafiltration tube, centrifuged at 10,000 g at 4°C for 30 min, and the permeate was collected, which w...
Embodiment 2
[0045] 2.1 Preparation of peptide samples
[0046] The experimental samples were subjected to protein brandford quantification or cell calculation quality control. Take an equal amount or volume of samples and add them to the peptide extraction lysate (8M urea / 100mM TEAB, pH 8.0, 1mMPMSF / 2mM EDTA), mix well and incubate on ice for 5min, add the final DTT with a concentration of 10mM was sonicated in an ice bath for 15min, then centrifuged at 13000g at 4°C for 30min, and the supernatant was transferred to a new centrifuge tube; DTT was added to the centrifuge tube to a final concentration of 10mM, and the reduction reaction was carried out in a water bath at 56°C for 30min to obtain Restore the sample. Subsequently, IAM was added to a final concentration of 55 mM, placed in the dark at room temperature for 30 min for alkylation reaction, and the initial sample was obtained, treated with a 10 kD ultrafiltration tube, centrifuged at 10,000 g at 4°C for 30 min, and the permeate wa...
Embodiment 3
[0058] 3.1 Preparation of peptide samples
[0059] The experimental samples were subjected to protein brandford quantification or cell calculation quality control. Take an equal or equal volume of samples and add them to peptide extraction lysate (8M urea / 100mM TEAB, pH 8.0, 1mM PMSF / 2mM EDTA), mix and incubate on ice for 5min, add DTT with a final concentration of 10mM was sonicated in an ice bath for 15min, then centrifuged at 13000g at 4°C for 30min, and the supernatant was transferred to a new centrifuge tube; DTT was added to the centrifuge tube to a final concentration of 10mM, and the reduction reaction was carried out in a water bath at 56°C for 30min. Get a reduced sample. Subsequently, IAM was added to a final concentration of 55 mM, placed in the dark at room temperature for 30 minutes for alkylation reaction, and the initial sample was obtained, treated with a 10kD ultrafiltration tube, centrifuged at 10000g, 4°C for 30 minutes, and the permeate was collected, whic...
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