Biology Reference
In-Depth Information
150 mM NaCl
280 mM sorbitol (182.19 g/mol)
pH range: 5.0-5.5
PMCB pH 6-7
Before adjusting the pH of the PMCB earlier, take a 50 mL aliquot (it should
be pH 7).
PAA (require 5 mL per sample)
1 mg/mL PAA in PMCB (pH 6-7)
First, prepare 50 mg/mL stock in water (dissolves slowly) and then dilute
accordingly.
Lysis buffer (prepare 50 mL for two samples)
2.5 mM imidazole, pH 7.0
10
g/mL aprotinin
m
g/mL leupeptin
PMSF and Na
3
VO
4
can be added as well
Ludox
Make 5% (v/v) mixture of ludox in PMCB (5 mL per sample).
Histodenz (Nycodenz)
Measure 10 g powder and add 5.5 mL lysis buffer.
Vortex repeatedly, takes
30-60 min to dissolve.
Take 7 mL of this and top up to 10 mL with lysis buffer.
This solution can be frozen as a stock, but ensure it completely dissolves upon
thawing.
4
10
m
Sample buffer (prepare 50 mL, can be stored at room temperature)
8% SDS
-Mercaptoethanol
250 mM Tris, pH 6.8
40% Glycerol (v/v)
2% Bromophenol blue
ddH
2
O
PBS, pH 7.4
8.0 g of NaCl
0.2 g of KCl
1.44 g of Na
2
HPO
4
0.24 g of KH
2
PO
4
Up to 1 L ddH
2
O
8%
b
22.2.1.3
Equipment
Optima XL-90 ultracentrifuge with SW40 rotor (Beckman) or similar
This protocol is designed for an 11 mL tube. Depending on the rotor used,
calculate the volume of lysate and other Histodenz layers accordingly so
that they fit in the tube. Smaller rotors (e.g., SW60, Beckman) may also be
used.
