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 don’t 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
0.2M solution contains 27.22g/l.
2
O, M.Wt. 136.09;
Acetic acid, glacial is ~17.47M. x ml 0.2M NaOAc and y
ml 0.2M HOAc mixed. pH, 18°C
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
Water 200ml
400ml
Filter this buffer through a 0.2
μ m 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
total volume
(0.2M) (0.2M)
5.5 442.5
μ l 57.5
μ l 500 μ l
5.0 350 μ l 150 μ l 500 μ l
4.5 215 μ l 285 μ l 500 μ l
Dilute each of these to 10ml in water, filter-sterilise through a 0.2
μ m filter.
Dilute your protein to e.g. 5 μ g/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
2.5ml
Water 2.5ml
10ml
Filter-sterilise through a 0.2
μ m filter.
Surface activation
Ensure the sample and buffer don’t 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.