His-Buster Cobalt Affinity Gel: Buffers and Method Recommendations

Recommended buffers

  1. Buffer substances – 25 mM Tris-HCl, pH 7.5-8.0, or HEPES-Na, pH 7.0-7.5, or Sodium Phosphate, pH 7.0-7.5 (it allow to work with different proteins far away from pI).
  2. Salt – NaCl or KCl not less 500 mM.
  3. Imidazole – 5 mM in sample and wash buffers. During washing imidazole concentration may be increased up to 15-20 mM (protein and pH dependent).
  4. Glycerol – 10% (v/v).
  5. Detergent – OGP, Triton, Tween, NP (0.05 – 0.1%).

For elution purposes we recommend to increase imidazole concentration up to 300-500 mM.

General guidelines for working with protein solutions:

Temperature: Unless indicated otherwise, try to keep protein solutions cold by placing them in an ice bucket or working in a cold room. This will slow down uncontrolled proteolytic degradation and other enzymatic reactions that could adversely affect the protein of interest. Also, most proteins are more stable at low temperatures. Some steps in purification protocols can be carried out at room temperature if a cold room is not available, and sometimes it is even desirable to carry them out at room temperature to accelerate the rates of certain reactions, but these cases are exceptional and will generally be noted.

Denaturation: Avoid foaming or introducing bubbles, because air-liquid interfaces can denature proteins. Some bubbles are unavoidable, but work in ways that minimize them. Avoid vortexing protein solutions, and when mixing by repeatedly suctioning and expelling solutions with a pipetman or seorological pipette, don´t suction or expel the solution completely from the pipette until you are done mixing. Excessive heat also promotes denaturation, so when using techniques such as sonication, homogenization or other heat-generating mechanical methods of cell disruption, keep containers on ice as much as possible.

Sterility: Protein solutions don´t need to be kept sterile unless their intended purpose requires it. However, for long term storage of liquid protein solutions at -20°C or 4°C (see next point), NaN3 (sodium azide) can be added to a final concentration of 0.01-0.05% to avoid bacterial growth.

Storage: For short term use, protein solutions are usually stable when kept on ice or at 4°C. For long term storage, most protein solutions can be snap-frozen in liquid nitrogen after addition of 10% glycerol and stored at -80°C. The idea behind adding glycerol is that when aqueous solutions of proteins containing salts are frozen, an ice crystal lattice may form first and exclude salt ions as well as proteins, resulting in a non-uniform distribution of salt with "pockets" of very high concentration, as well as dehydration of the protein molecules, both of which could potentially denature proteins. Glycerol interferes with the formation of the ice crystal lattice, resulting in a more even distribution of all species in the solution. Along these same lines, snap-freezing in liquid nitrogen and quickly thawing at 37°C is better than slow freezing or thawing, which would allow more time for segregation of the different species in the solution. Many proteins are also stable at -20°C in a solution kept liquid by the presence of 50% glycerol. To make a 50% glycerol solution of protein, instead of adding glycerol directly (which would result in unnecessary dilution and would be hard to mix), the protein sample can be dialyzed against a solution of 50% glycerol prepared in the desired storage buffer. This procedure actually concentrates the protein significantly (about 5-fold or more) owing to the rapid osmotic efflux of water from the dialysis bag. This allows for subsequent dilution of the glycerol to acceptable concentrations for biological assays without significant dilution of the protein itself.

HIS-Buster Nickel Affinity Gel is compatible with the following compounds.

(At least at the concentrations given.)
Reducing agents 
20 mM 2-mercaptoethanol
5 mM TCEP (Tris[2-carboxyethyl]phosphine)
10 mM reduced glutathione
Denaturing agents 
8 M urea
6 M guanidinium hydrochloride
2% Triton X-100
2% Tween 20
2% NP-40
2% n-Octyl-Beta-D-Glucopyranoside
Buffers substances 
50 mM sodium phosphate, pH 6.5-7.5
50 mM HEPES-Na, pH 7.0-8.0
50 mM Tris-HCl, pH 7.5-8.5
50 mM citrate
Other additives 
500 mM imidazole
30% ethanol
20% isopropanol
50% glycerol
50% ethylene glycol
100 mM sodium sulfate
2 M NaCl

For a more detailed description of methods and buffers, please visit our online shop to download a purification protocol specific to your product(s).