Biotinylation Kit (Sulfo-OSu)BK01 Product Manual

 The system using avidin-biotin complex is being used widely in the field of immunological approch such as EIA (enzyme immunoassay) or immunohistochemistry.Biotin can be labeled without loss of activity to proteins, antibodies and enzymes. And avidin has a strong affinity (Kd～10^-15 mol/l) with biotin.
 Biotin-(AC₅)₂Sulfo-OSu, the biotinylation reagent in this kit, has a water-soluble activated ester and a long spacer in its structure. Therefore, it is possible to label amino groups of proteins.

The reaction of Biotin-(AC₅)₂Sulfo-OSu with a protein

Biotin-(AC5)2Sulfo-OSu	10mg x 4 vials
Sodium bicarbonate powder	4 bottles
Approximately 50 mmol/l of carbonate buffer can be prepared by adding 10ml of water to the bottle.
Gel filtration column	4 pieces
PBS tablet	4 tablets
10 mmol/l-PBS buffer can be prepared by dissolving a tablet with 100ml of water
Sample tube	8 tubes

• Purification of labeled protein is possible with this kit alone.
• Labeling of four different protein is possible.
• It doesn’t need to weigh the reagents. here is no deterioration of Biotin-(AC5)2Sulfo-OSu upon repeated unsealing.
• It doesn't need to use an organic solvent such as DMF or DMSO because of its water-solublility.
• The rate of biotin-labeling can be controlled by changing the amount of Biotin-(AC5)2Sulfo-OSu.Labeling of 1-5 mg of protein with Biotin-(AC5)2 Sulfo-OSu is possible using one vial.

1. Add 10 ml of deionized or distilled water to Sodium bicarbonate powder in the bottle to dissolve.  (Preparation of carbonate buffer)
2. Add 1-5 mg of protein to the sample tube(record the actual weight accurately.)
3. Add 0.5 ml of carbonate buffer to this sample tube. After the tube is capped, protein is dissolved using vortex.
4. Dissolve Biotin-(AC₅)₂Sulfo-OSu in a tube with 0.75 ml of deionized or distilled water.
   • Step 4. and 5. should be carried out quickly because the reagent has a chance to be hydrolyzed.
5. Add the above Biotin-(AC₅)₂Sulfo-OSu solution to the protein solution in 3) according to the following table.
   
   Example: 12.5 ml of Biotin-(AC₅)₂Sulfo-OSu solution is added to 0.5 ml of Protein A solution (1 mg/ 0.5ml)  when 10 equivalent of Biotin-(AC₅)₂Sulfo-OSu is reacted with Protein A. On other hand, when 2 equivalent of Biotin-(AC₅)₂Sulfo-OSu is reacted, 2.5 ml of Biotin-(AC₅)₂Sulfo-OSu solution is added.)
6. After being capped, the tube is mixed using vortex and then incubated at 25℃ for 2.0 hrs.
7. Purify the protein by using the gel filtration column.

   a) A PBS tablet is dissolved with 100 ml of deionized or distilled water using a 100 ml-volumetric flask.

   b) The upper cap of the gel filtration column is removed, and the liquid inside is thrown away.

   c) The drain cap is removed, and load 10 ml of PBS with pipette to equilibrate.

   d) Load 500 ml of the labeled protein solution to the column.

   e) Put 2 ml-sample tube under the drain, and load 1 ml of PBS to the column to elute the biotinylated protein.

 

The final concentration of the biotinylated protein (A) is calculated from the following equation.

 

A (mol/l) = (X g /MW Protein) × (1000ml / Y ml) × 0. 5 

  X: weight of protein

  Y: volume of carbonate buffer to dissolve protein

 

• Store at 0-5℃
• Prepare the Biotin-(AC₅)₂Sulfo-OSu solution before use. Biotin-(AC₅)₂Sulfo-OSu will be readily hydrolyzed. Avoid storing the solution as stock.
• Keep the labeled protein at 0-5℃. Add 0.1%-sodium azide as a preservative.

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