Protein collection and prep for proteomics (Ostracods)

Protein collection

1. Place ostracods in collection chamber
2. Shock for 10secs with 12V or until bioluminescent response is elicited (note: do not shock continuously for over 10secs, instead use short shocking pulses)
3. Remove fluid using a pipette and place into sterile 1.5mL tube. If possible, wear gloves and clean work station of dust or other contaminants.

Protein precipitation

1. Add 100% trichloroacetic acid (TCA) to bring solution to 20% TCA
2. Leave at -20*C overnight
3. Spin down at max speed for 10min at 4*C
4. Wash (x3 times) with an ice cold solution of 10% 0.01 M HCl and 90% acetone
5. Air dry pellet for 10min (usually)
6. Resuspend in ~80ul of 50% 100mM TRIS (pH 8.5), 50% 8M Urea

Protein digestion=

1. Add 100mM TCEP to a final concentration of 5 mM. Incubate at room temperature for 5min.
2. Add 500 mM Iodoacetamide to a final concentration of 10 mM. Make fresh each time. Incubate at room temperature for 15min in the dark.
3. Dilute samples by a factor of four with 100 mM TRIS-HCl (pH 8.5)
4. Add 100 mM CaCl2 to final concentration of 1 mM
5. Add 1ul (0.5 ug/ul) trypsin
6. Incubate overnight at 37*C in the dark