Peptide isolation and purification experiments for protein internal sequence analysis
Peptide isolation and purification experiments for protein internal sequence analysis
One of the most important aspects of molecular biology is the analysis of trace proteins, making possible the cloning of cDNA sequences encoding the proteins to be studied. To do this efficiently, it is often necessary to know the internal sequence of the protein. In this respect, the efficient isolation of peptides is extremely important. This experiment was derived from the Laboratory Guide for Protein Purification and Characterization by Houzhu Zhu.
Operation method
Peptide isolation and purification experiments for protein internal sequence analysis
Materials and Instruments
Calomas Brilliant Blue G Acetic Acid Methanol Achromobacter Trypsin I Trifluoroacetic Acid Acetonitrile 2-Propanol Move Materials and equipment For more product details, please visit Aladdin Scientific website.
SpeedVac Rotary Concentrator Tween-20 UltrafreeTM MC Filter C18 Reversed Phase HPLC Columns
Caulmers Brilliant Blue G (0.05% acetic acid-20% methanol solution)
Acetic acid (5% of 10% methanol solution)
Methanol (50% in water and 10% in water)
SpeedVac Model Rotary Concentrator (Savant Instruments, Inc.)
Achromobacter trypsin I [lysyl endopeptidase; 50ng/ul of 0.1mol/LTri-HCl (pH9.0)
Tween-20 [0.1% of 0.1mol/L Tris-HCl (pH9.0) solution].
UltrafreeTM MC filter (22um; Millipore Corp.)
Trifluoroacetic acid (TFA; 0.1% in 50% acetonitrile)
C18 reversed-phase HPLC column (2.1X250 mm; 5um, 300A) (VYDAC)
Acetonitrile
2-Propanol
Operating Procedures
1) Separate the proteins to be studied by SDS-PAGE.
2) Stain the gel with 0.05% Coomassie G for 15~30 min.
3) Decolorize the gel with 10% methanol solution containing 5% acetic acid.
4) Soak in water for 10 min.
5) Cut off the protein band from the gel and transfer to a microcentrifuge tube. Cut off a section of protein-free gel as a control. If necessary, the gel can be cut into smaller pieces so that it can be easily sunk to the bottom of the microcentrifuge tube.
6) Add 1 ml of 50% methanol to each tube. Incubate for 20 min at room temperature and discard the supernatant.
7) Add 1 ml of 10% methanol to each tube. Incubate for 20 min at room temperature, discard supernatant.
8) Dry gel pieces in a SpeedVac rotary concentrator for 2 min.
9) Add 10ul of Achromobacter Protease I to each tube.
10) Add a minimum volume of 0.1 mol/L Tris-HCl (pH 9.0) containing 0.1% Tween-20 to each tube - just enough to submerge the gel fragments.
Note: Gel fragments will swell, so it is important to ensure that they remain submerged in the descaler buffer.
11) Incubate each tube at 30°C for 24 hours.
12) Centrifuge tubes in a microcentrifuge and transfer the supernatant from each tube to a 22-um Ultrafree MC filter. centrifuge at 12,000 r/min for 2 min. retain the filtrate.
13) Add 1 volume of 50% acetonitrile solution containing 0.1% TFA to the gel fragments left in each tube-enough to submerge the gel fragments Incubate at 4°C for 30 min.
14) Repeat steps 12 and 13. Combine filtrates from each sample.
15) Spin the sample in a SpeedVac rotary concentrator to a volume of less than 100 ml. Add the appropriate amount of HPLC equilibrium to make a sample for HPLC analysis.
16) Separate the peptides in the digest with a reversed-phase HPLC C18 column. The peptides were eluted with a gradient of acetonitrile/2-propanol (3:1) containing 0.09% TFA. The UV absorption of the peptides was detected at 214 mn (for peptide bonds) and 295 nm (for tryptophan).