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General Protocol for Protein Labeling Using NHS and Maleimide Fluorescent Dyes

Labeling of Proteins Using NHS-Ester Fluorescent Dyes


General protocol for labeling of proteins including Protein A, Streptavidin, IgG and other antibody types with maleimide and NHS-ester dyes.

  1. Dissolve 1 mg of NHS ester fluorescent dye in 50µl absolute, amine free DMF to reach a final concentration of approx. 25nmol/µl).
  2. Dissolve the desired amount of protein in 50mM bicarbonate buffer, pH 9.0. For optimal labeling the protein concentration should usually be between 5-20 mg/ml. Concentrations lower than 2mg/ml will decrease the efficiency of the reaction. Do not use solutions or buffers containing amine substances such as Tris, glycine or ammonium ions.
  3. Transfer an appropriate volume of the NHS ester stock prepared in step 1 above dropwise under stirring to the protein solution. For best results it is recommended to add an equimolar amount or the double excess of label to the protein to obtain a dye to protein ratio between 1 and 2. Higher molar excesses of the label can lead to overlabeling of the protein causing a decrease in quantum yield of the conjugate.
  4. Incubate for 1 hour at room temperature.
  5. Separate labeled protein from unreacted dye using a Sephadex column (Sephadex G25 medium) operated with 100mM PBS, pH 7.5. Alternatively, BioGel P-30 or equivalent gel filtration media, equilibrated with a buffer of your choice, may be used. The first running coloured band is the fluorescent dye-labeled protein.
  6. Generally proteins labelled with fluorescent dyes should be stored under the same conditions used for the unlabeled protein. To preserve the samples sodium azide should be added to a final concentration of 2mM.

Labeling of Proteins Using Maleimide Fluorescent Dyes


A general procedure suitable for conjugation of thiol-reactive probes, e.g maleimides, to proteins is outlined below.

  1. Dissolve protein at a concentration of 50-100 µM in a suitable buffer at pH 7.0-7.5 (e.g. 10-100 mM phosphate, Tris or HEPES) at room temperature. In this pH range, the protein thiol groups are sufficiently nuclephilic so that they react almost exclusively with the reactive dye in the presence of the more prominent amines present in the protein, which are protonated and relatively unreactive.
  2. At this stage reduction of disulfide bonds in the protein is performed by the addition of a 10-fold molar excess of a reducing agent such as DTT or TCEP. Please note that if DTT is used, dialysis is required to remove excess DTT prior to addition of the reactive dye. Removal of TCEP is not required during conjugation with iodoacetamides or maleimides.
  3. Thiol modifications are best performed in an oxygen-free environment to prevent oxidation of the free thiols to disulfides. This is particularly important if the protein has been treated with a reagent such as DTT prior to thiol modification. To prevent oxidation of thiols to disulfides all buffers should be deoxygenated and the reactions carried out under an inert atmosphere.
  4. Prepare a 1-10 mM stock solution of the reactive dye in a suitable solvent immediately before use. All stock solution should be protected from light as much as possible.
  5. Add 10-20 moles of reactive dye for each mole of protein drop-wise to the protein solution as it is stirring.
  6. Incubate the mixture for 2 hours at room temperature or overnight at 4°C. For maleimides, it is essential to protect the reaction mixture from light as much as possible.
  7. After incubation an excess of glutathione, mercaptoethanol, or other soluble low molecular weight thiol can be added to consume excess thiol-reactive reagent, thus ensuring that no reactive species are present during the purification step.
  8. Purify the final conjugate on a suitable gel filtration column such as a Sephadex G-25 column, or by extensive dialysis at 4°C in an appropriate conjugate storage buffer.
  9. Generally proteins labelled with fluorescent dyes should be stored under the same conditions used for the unlabeled protein. To preserve the samples sodium azide should be added to a final concentration of 2mM.

Note: Maleimides are not very stable in solution of any kind and should thus not be stored for more than 24 hours in solution. DMSO is a suitable solvent for resuspension of the reactive dye in most cases. Maleimides are also sensitive to light and should be protected from light at all times.

Buffers


100mM Phosphate Buffered Saline (PBS), pH 7.5 (1 liter)
  1. Dissolve 3.17 g of NaH2PO4 • H2O and 13.7 g of Na2HPO4 • 2 H2O in 800 ml of double distilled.
  2. Adjust the pH to 7.5 with 1 M HCl or 1 M NaOH.
  3. Add double distilled water to a final volume of 1 liter.

50mM Bicarbonate buffer, pH 9.0 (500 ml)


Use this buffer to dissolve the protein to be labeled with the Cyto NHS-ester dye.

  1. Dissolve 2.1 g of NaHCO3 in 400 ml of double distilled water.
  2. Adjust pH to 9.0 with 1 M HCl or 1 M NaOH.
  3. Add double distilled water to a final volume of 500 ml.