Nucleotide separation
in
phosphate free
buffer
Phosphate - the problem
Phosphate
+
(any concentration between 5-100 mM;
but small volumes necessary)
Acetonitrile
(75-100%, maybe less)
Phosphate crystals!
Atlas of Canine and Feline Cytology, Saunders, 2001
Phosphate - the problem
Phosphate
Phosphate Acetonitrile
Acetonitrile
now flow
HPLC
HPLC - a solution
Nucleotide separation
in
phosphate free
buffer
HPLC - a solution
Reference: 0.1 M phosphate; pH 5.5-6.5; ACE C18 AR, 2.1 mm×15 cm,
GTP/GDP: no separation; AMP/ADP/ATP: weak separation
0.1 M MES; pH 5.5: better than phosphate; still bad
0.1 M MES; pH 5.5 + 2% methanol: better than phosphate; still bad
0.1 M MES; pH 5.5 + 6% methanol: like phosphate
0.1 M HEPES; pH 7.5: like phosphate
0.1 M triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile:
good resolution and separation (excellent for GTP/GDP)
Triethylammonium bicarbonate buffer
0.1 M triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile
GTP
GDP
0.1 M phosphate, pH 6.0
(ACE C18 AR, 2.1 mm×25 cm
column)
GDP
GTP
100 µM GTP, 100 µM GDP
in 8 mM PIPES pH 6.8
GDP alone
GTP alone
0.1 M triethylammonium
bicarbonate/acetate pH 7.4,
2% acetonitrile (ACE C18
AR, 2.1 mm×15 cm column)
Triethylammonium bicarbonate buffer
0.1 M triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile
Expensive (48$ per l)
Lower concentration (35 mM)
Triethylammonium bicarbonate buffer
35 mM triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile
100 µM GTP, 100 µM GDP
expensive (48$ per
in 8l) mM PIPES pH 6.8
lower concentration (35 mM)
Triethylammonium bicarbonate buffer
A real experiment (GTP and GDP in tubulin from Cytoskeleton.com)
GTP
45%
GDP
55%
0.1 M phosphate, pH 6.0
(25 cm column)
?
35 mM triethylammonium
bicarbonate/acetate pH 7.4,
2% acetonitrile (15 cm
column)
?
GDP
GTP
19-22% 78-81%
?
Peaks are not
really nice,
because of the
low pH of the
injected sample
Triethylammonium bicarbonate buffer
It also works with AMP/ADP/ATP
AMP
ADP
ATP
AMP
ADP
ATP
[Acetonitrile] (3.5-4% ?)
Triethylammonium bicarbonate buffer
Does not form salts in Good buffers (MES, HEPES, PIPES) or water
Can be mixed with acetonitrile, isopropanol
Can be mixed with 5 mM Mg2+ (I have not tried Ca2+)
Triethylammonium bicarbonate buffer
Limitations
HCO3-
CO2
+ OH- (pH!)
Triethylammonium bicarbonate buffer
Limitations
1) Cool the buffer (4°C)
2) Make fresh buffer every day
Triethylammonium bicarbonate buffer
35 mM triethylammonium bicarbonate/acetate pH 7.4, 2% acetonitrile
Addition of 80mM HEPES pH 7.4 results in a more stable pH
But there is no good nucleotide separation anymore
Triethylammonium bicarbonate buffer
A short manual
1) 86-95% water (4°C)
2) Add 3-10% triethylammonium bicarbonate buffer (1 M) and 2-4%
acetonitrile
3) pH to 7.4 with acetate (7.1 when you need more than 6h for your
experiments)
Always store on ice; the pH of the buffer should be ≤8.0
Keep in mind that substances in your samples might bind to the column;
include the one or the other cleaning step between your experiments when
you have many samples