Analysis of Digested Protein using HPLC-UV

Analysis of Digested Protein using HPLC-UV

Sample:

  1. Insulin
  2. BSA
  3. Mab
  4. Thyroglobin

HPLC Analytical Conditions:

Column: Develosil HSR C18 Peptide, 3 µm ( 4.6 x 75 mm)
Mobile Phase:A) Water + 0.1% TFA
B) Acetonitrile +0.1% TFA
Gradient:Further details here
Flow Rate:1.0 mL/min
Temperature:50°C
Detection:UV at 210 nm
Injection Volume:(1) 10 µL
(2)-(4) 20 µL

Protein Digestion:

  1. Weight 1.0 to 2.0 mg each of insulin, BSA, monoclonal antibody, and thyroglobulin.
  2. Add 1.0 mL of SMART digest buffer and stir thoroughly.
  3. Place 200 µL of the above sample in a SMART digest tube and mix at a T=70°C, 1,400 RPM 2 hours using Thermomixer C.
  4. Centrifuge the prepared, mixed samples for injection.

The “SMART Digest kit” used in this research is a registered product of Thermo Fisher SCIENTIFIC.

The “Thermomixer C” used in this research is a registered product of Eppendorf.

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