Acetate buffers for amine coupling of proteins to Biacore sensor chip surfaces.

N.B. for amine coupling of proteins to e.g. CM5 chips, ensure the sample and buffer
dont contain amines e.g. buffer components (Tris), BSA (used to protect dilute
proteins)

Sodium acetate-acetic acid buffer solutions, pH 3.7-5.6
Sodium acetate trihydrate, CH
3
COONa.3H
2
O, M.Wt. 136.09;
0.2M solution contains 27.22g/l.

Acetic acid, glacial is ~17.47M.

x ml 0.2M NaOAc and y ml 0.2M HOAc mixed.
pH, 18C

X mL
0.2M NaOAc
Y mL
0.2M HOAc
3.7 10.0 90.0
3.8 12.0 88.0
4.0 18.0 82.0
4.2 26.5 73.5
4.4 37.0 63.0
4.6 49.0 51.0
4.8 59.0 41.0
5.0 70.0 30.0
5.2 79.0 21.0
5.4 86.0 14.0
5.6 91.0 9.0

Source; Data for Biochemical Research, 3
rd
edition. R.M.C. Dawson, D.C. Elliott,
W.H. Elliott & K.M. Jones, Oxford Science Publications, 1986.

For amine coupling, the system can be primed with 100mM acetate, e.g. pH 5.6.

Stock solutions
Sodium acetate, anhydrous; 82.04 x 0.2M x 0.25l = 4.102g, dissolved in 250ml water.
Acetic acid, glacial; 0.2M/17.47 = 2.86ml, diluted in 250ml water.

Running buffer
To prepare 0.1M acetate running buffer pH 5.6;
Sodium acetate 0.2M 182ml
Acetic acid, 0.2M 18ml
Water 200ml
400ml

Filter this buffer through a 0.2m filter.
Run prime3.blm after docking a CM5 sensor chip, to flush the system thoroughly
with the new buffer.

Immobilisation buffers
For amine coupling, low ionic strength, acetate buffers covering a range of pHs are
usually tested for optimum non-specific binding to the chip surface. This pre-
concentration traps the protein on the surface for a long time to maximise the
chances of the protein reacting with an NHS/EDC-activated site. The optimum pH is
often just below the isoelectric point (pI) for the protein.
An estimate of the pI of your protein can be obtained by pasting the amino acid
sequence (plus any affinity tags) at the Expasy web-site;
http://www.expasy.ch/tools/pi_tool.html

pH sodium acetate
(0.2M)
acetic acid
(0.2M)
total volume

5.5 442.5l 57.5l 500l
5.0 350l 150l 500l
4.5 215l 285l 500l
Dilute each of these to 10ml in water, filter-sterilise through a 0.2m filter.

Dilute your protein to e.g. 5g/ml initially, into these buffers and perform manual
injections. Find a condition which gives you the required level of binding at a slow
enough rate that you can stop the injection when the required level has been reached.
For good kinetic analysis, you may have to use lower protein concentrations; for
ligand fishing or analysis of low molecular weight analytes, you might use higher
protein concentrations, longer injection times or a lower immobilisation buffer pH.

Elution buffer
Non-specifically bound protein should be stripped off using a high salt buffer between
injections, e.g. 1) acetate buffer 0.1M/NaCl 1.0M see below; 2) PBS containing
1.0M NaCl; 3) HCl 5mM.

Sodium acetate, 0.2M 4.55ml
Acetic acid, 0.2M 0.45ml
NaCl, 4M 2.5ml
Water 2.5ml
10ml
Filter-sterilise through a 0.2m filter.

Surface activation
Ensure the sample and buffer dont contain amines e.g. buffer components (Tris),
BSA (used to protect dilute proteins)

Activate the surface using freshly-mixed NHS and EDC solutions once you have
determined the optimum protein concentration and pH.

Inject protein at the required concentration and pH to give the required level of
immobilisation. Send the protein to waste when the required level has been reached. If
you are immobilising a small protein, you may want to overshoot, as coupling can be
less efficient. Large proteins are coupled more efficiently.

Use a high salt injection to remove any unbound protein. Avoid amine containing
buffers or samples.

Block unreacted, activated sites with ethanolamine (1.0M) using an injection of the
same length as the NHS/EDC injection.
As an alternative to ethanolamine from Biacore, purchase the best quality
ethanolamine and dilute to 1.0M.
Sigma
411000-100ml.
M.Wt. 61.08; density = 1.012g/ml, i.e. 1012g/litre

Therefore concentration = 1012/61.08 = 16.57M.

For a 1M solution; 1/16.57 x 10ml = 604ul diluted to 10ml.
Vortex thoroughly to mix.