Biology Reference
In-Depth Information
2
Materials
Prepare all solutions using ultrapure water and reagents with
highest purity or grade. Use low-binding tips and tubes to mini-
mize peptide loss caused by adsorption to the plastic, but do not
use surface coating materials (e.g., siliconized) which may
contaminate samples. Take maximum care to avoid keratin
contamination.
2.1 Protein
Extraction
and Digestion
1. Mortar and pestle, or bead mill.
2. Liquid nitrogen.
3. Tris buffer: 10 mM Tris-HCl, pH 9.0. Store at 4 °C.
4. Extraction buffer (Prepare just before use): 8 M Urea in Tris
buffer, phosphatase inhibitors (
see
Note 1
). For 100 ml buffer
preparation, weigh 48.048 g of urea and add the Tris buffer to
a fi nal volume of 100 ml. Add phosphatase inhibitors.
5. Protein quantifi cation reagents (e.g., BCA Protein Assay Kit;
Pierce).
6. Ammonium bicarbonate (NH
4
HCO
3
) (Prepare just before
use): For 100 ml of 50 mM buffer, weigh 0.3953 g of
NH
4
HCO
3
and dissolve in 100 ml water.
7. Dithiothreitol (DTT): Dissolve DTT in water to a concentra-
tion of 1 M. Divide into small aliquots and store at −20 °C.
8. Reduction buffer (Prepare just before use): 10 mM DTT in
the 50 mM NH
4
HCO
3
. Dilute the 1 M DTT with the 50 mM
NH
4
HCO
3
.
9. Iodoacetamide (IAM): Dissolve IAM in water to a concentra-
tion of 1 M. Divide into small aliquots and store at −20 °C.
10. Alkylation buffer (Prepare just before use): Dilute the 1 M
IAM with 50 mM NH
4
HCO
3
to a fi nal concentration of
50 mM IAM.
11. Lysyl Endopeptidase (LysC), MS grade.
12. Trypsin, MS grade.
2.2 StageTip Column
and Spin Adaptor
Preparation
Some types of StageTips and spin adaptors are commercially
available.
1. Empore C18 extraction disk (Part Number 2215, 3 M).
2. Empore C8 extraction disk (Part Number 2214, 3 M).
3. Methanol.
4. 16 gauge, Kel-F Hub NDL, 2 in., point style 3 (Part Number
90516, Hamilton) needle to punch out small disks for prepar-
ing 200
μ
l pipet tip used in StageTip columns.
