Biomedical Engineering Reference
In-Depth Information
Lare
loaded, no washing step is required, samples are loaded
onto the beads, eluted by centrifugation, and the column
is directly loaded again.
18. Column storage: For short-term storage, after equilibra-
tion centrifuge the column at 2000
depletion step (
see
Steps 8-15). If more than 100
μ
×
g
for 30 s, apply the
bottom plug and add 300
L of 1X Equilibration buffer.
For long-term storage prepare the storage solution: 10
μ
L
Proteoprep Preservative Concentrate in 5 mL of 1X Equi-
libration Buffer. 300
μ
L is added as for short-term storage.
Columns are stored at 4
◦
C.
μ
3.4.BufferExchange
Both concentrated fractions (i.e. the depleted-low-abundance and
the bound-high-abundance fractions) require buffer exchange
prior to digestion with trypsin. Several buffers are suitable for the
trypsin digestion step, such as 50 mM ammonium bicarbonate
buffer. We use 0.5 M sodium carbonate solution, pH 8.5.
1. Both concentrated fractions are diluted in 2 mL 0.5 M
sodium carbonate solution and concentrated using Microsep
1 K Centrifugal Devices as above.
3.5.Protein
Quantification
1. Protein concentration is determined using Bio-Rad Protein
Assay Reagent (Bio-Rad). For the standard curve known
concentrations of albumin (1-10
μ
μ
L) are used.
2. The low concentration range assay is used in the test tube
format. 2
g/
μ
L of standard or sample is added to 798
μ
Lof
L of Bio-Rad reagent is added, mixed,
and incubated for 10 min at room temperature.
3. The absorbance at the wavelength of 595 nm is measured in
a spectrophotometer.
MilliQ water. 200
μ
The protocol is based on the Mini-Protean System (Bio-Rad,
Hemel Hempstead, UK).
1. Clean the glass plates to be used and assemble the gel plates
in the gel casting unit.
2. Prepare the 10% separating gel by mixing 4 mL MilliQ
water, 3.3 mL 30% acrylamide mix, 2.5 mL 1.5 M
Tris/HCl (pH 8.8), 100
3.6.QualityControl-
SDS-Polyacrylamide
GelElectrophoresis
(SDS-PAGE)
μ
L 10% SDS, 100
μ
L 10% ammo-
nium persulphate, and 4
L TEMED. Directly after addi-
tion of TEMED pour the gel and leave sufficient space for
the stacking gel. Overlay the gel carefully with 0.1 % SDS
solution. After polymerisation (
μ
30 min) discard the 0.1 %
SDS solution, rinse with MilliQ water, and air dry.
3. Prepare the stacking gel by mixing 2.7 mL MilliQ water,
670
∼
μ
L 30% acrylamide mix, 500
μ
L1MTris/HCl(pH
6.8), 40
μ
L 10% SDS, 40
μ
L 10 % ammonium persulphate,
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