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- Lysed Hek Cell (cell lysed in 6M Guanidine Hydrochloride in 100 mM Tris-HCl pH8.0 followed by heating at 95C for 5 min., determine the total protein concentration by Bradford assay.
- Aliquot 2 1 mg of the above protein sample.
- Reduce disulfide bonds and carbamidomethylate cysteine residues by adding a 1:10 volume of reduction/alkylation buffer (100 mM TCEP and 400 mM 2-chloroacetamide) to the samples. Incubate samples for 5 min at 45 C with shaking (1,500 rpm).
- Diluted (DF=3) above samples with 100 mM Tris-HCl pH8.
- Add 10 ul of LysC (2 ug/ul) and 40 ul of Trypsin (0.5 ug/ul) at an enzyme-to-substrate ratio of 1:100 (wt/wt) (e.g., 2 ug of each enzyme for 200 ug of protein), reseal, and digest overnight at 37C with shaking (1,500 rpm).
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- Beads plate: Aliquot proper amount of beads (PureCure or 250 ul, IMAC 75 ul) to the corresponding position of the standard plate
- Peptide plate (Standard 96 well plate): Resuspend peptide samples in 0.1% TFA in 80% ACN. Aliquot appropriate amount of peptide to the corresponding position of the standard plate.
- Three wash plates (Standard 96 well plate): Aliquot 150 ul of 0.1% TFA in 80% ACN to the corresponding positions.
- Elution plate: Freshly prepare elution buffer (50:50 Acetonitrile: (1:20) ammonium hydroxide/water). Add 50 ul of the elution buffer to the corresponding position to the standard 96 well plate when the KingFisherFlex paused.
- BindIt program used at KingFisherFlex: IMAC_20200303.bdz
- After the entire procedure is done, transfer the total amount of the sample from the elution plate to another labeled tube contained 30 ul of 10% FA in 75% ACN solution. Mixed well.
- Transfer the above peptide mix to a Nest C18 column under the sample column set. Label the final sample tube. Centrifuge with 3000 rpm for 1 min. Dried samples by Speed Vac.
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