The Alexa Fluor® 647 Protein Labeling Kit is designed to allow you to quickly and easily label a protein with Alexa 647. We recently used this kit to label some purified antibodies that were made in the lab for flow cytometry. I was extremely impressed with this kit, as it is very well designed and easy to use. This kit includes everything needed to efficiently label and purify three different 1 mg samples of protein. The kit includes Alexa 647 lyopholized in three individual tubes (which conveniently contain mini stir bars), purification resin and columns to remove the labeled protein from free dye, sodium bicarbonate powder for buffering and a 10X stock of elution buffer. From start to finish, the protocol takes approximately 2-3 hours, but very little of this time is actually hands on.
The protocol calls for labeling 1 mg of protein in a 500 ul volume (2 mg/ml stock). Briefly, sodium bicarbonate is added to the protein solution (should be in PBS) and the sample is transferred to the provided tube containing the dye and stir bar. The protein/dye solution is incubated for 1 hour at room temperature while stirring on a magnetic stir plate. During this time, the column is packed by simply transferring the resin to the provided column and allowing it to drip through. After the labeling is complete, the solution is transferred to the packed column and allowed to enter the resin. The column purification is used to separate the conjugated protein from free dye. This kit employs size-exclusion resin – the larger protein-dye conjugate is too large to enter the resin and runs faster than the free dye, which must travel farther through the pores in the resin. Once the entire solution has entered the column, elution buffer is added until the labeled protein elutes. As the column drains, a blue band of the labeled protein separates from a blue band of the free dye. After 30 to 60 minutes, you collect the labeled protein as it drips from the column. Typically, the protein elutes in a volume similar to the volume added to the column. The absorbance of the solution is then measured to determine the concentration of the protein and the degree of labeling.
The Alexa Fluor® 647 Protein Labeling Kit provides an easy and fast way to efficiently label a protein with a fluorescent dye. These kits can be ordered with several different fluorophores, so you are not limited to Alexa 647. The only minor concern about this kit is that it recommends the protein to be rather concentrated, at 2 mg/ml (1 mg of protein in 500 ul). However, we were successfully able to label and purify protein at a concentration of 1 mg/ml (1 mg of protein in 1 ml instead of 500 ul). Even with this increase in volume, the purification column was long enough to separate the conjugated protein from the free dye.