Biotin Labeling Kit (50 KD Filtration Tube)

Structural formula
Molecular weight	341.38

Labeling Principle

Within a certain pH range, NHS-Biotin specifically reacts with primary amino groups (N-terminal and lysine residue side chains) to form a stable amide bond, so as to realize the coupling of NHS-Biotin with protein.

Materials Not Supplied

1. Pipettor and tips (0.5-10μL, 2-20μL, 20-200μL, 200-1000μL).
2. 37°C incubator.
3. Centrifuge (centrifugal force up to 12,000×g).

Experimental Operation

Experiment preparation

1. Read the instructions carefully.
   
2. Bring all reagents to room temperature for 20 min before use (note: the reagent components are temporarily unused are still in the refrigerator).
3. Infiltration of ultrafiltration tube: Add 500 μL of Labeling Buffer I into the dry filter device, stand at room temperature for 10 min, and discard Labeling Buffer I before adding the reagent to be labeled (the filter device should remain moist throughout the labeling process).
4. Preparation of NHS-Biotin: Dissolve 0.1 mg of NHS-Biotin NHS ester with 30 μL of DMF, and stand for 10 min until it is fully dissolved. At this time, the concentration of NHS-Biotin is 10 mM, and cover the tube for later use.

Labeling Process

Labeling procedure (This procedure is used to label 1 mg protein)

1. Concentration & buffer exchange: Put the filter device in the collection tube, add 1 mg of protein to be labeled into the filter device, add Labeling Buffer I to the final volume of 0.5 mL, cover the filter tube, centrifuge at 12,000×g for 5 min, and discard the liquid in the collection tube.
   Note:
   a) The maximum volume of the filter device is 0.5 mL.
   b) If the volume of 1 mg protein is greater than 0.5 mL, please add it in several times and concentrate it by centrifugation and ultrafiltration.
   c) If the protein to be labeled contains free amino groups (Tris, amino acids or other interferents, repeat ultrafiltration with Labeling Buffer I to ensure that it is removed fully).
2. Recovery & quantification: Invert the filter device into the collection tube, centrifuge at 1000×g for 2 min, collect the protein in the collection tube, take out the filter device, add an appropriate amount of Labeling Buffer II into the collection tube, make sure that the protein concentration is about 2 mg/mL. At the same time, add 0.5 mL Labeling Buffer II into the filter device and put it on a pipe rack for later use.
3. Labeling reaction: Immediately add 13.3 μL of 10 mM Elab Fluor® 488 to the protein solution, gently blow and mix fully, sealed with a lid, and incubate at 37°C for 30 min in the dark.
   
4. Blocking: Add 1 M Tris (pH 8.7) to stop the reaction at the ratio of 10 μL of 1 M Tris (pH 8.7) per 100 μg protein, mix fully and incubate at room temperature for 10 min.
5. Ultrafiltration & purification: Add an appropriate amount of 1×PBS into the above reaction solution to the final volume of 0.5 mL, gently mix and transfer the reaction solution to the filter device, make sure that the Labeling Buffer I in the filter device in step 2 should be discard (if the above reaction solution exceeds 0.5 mL, it can be transferred to the spin-dried filter device for several times after ultrafiltration), and cover the cap after matching with the collection tube, and centrifuge for 5~10 min at the speed of 12,000×g. Discard the liquid in the collection tube, replenish 1×PBS to 500 μL in the filter device, and repeat the centrifugal ultrafiltration operation for 2~3 times.
6. Collect labeled product: Add 0.2 mL 1×PBS into the filter device and pipet gently. Invert the filter device in another collection tube and centrifuge at 1000×g for 2 min. Collect the solution in the collection tube, which is the protein labeled by NHS-Biotin.

The storage and use of protein

Add 0.05~0.2% Proclin 300 or 0.05% sodium azide and stabilizer protein (such as 0.1% BSA) to the labeled protein, the protein can be stored at 2~8°C in the dark for 6 months. Or add the same volume of glycerol, the protein can be stored at -20°C for 6 months.

Storage

The unopened kit can be stored at 2~8°C for 1 year, and the dissolved NHS-Biotin can be stored at -20°C or -80°C for 1 week.

Notes:

1. Please select the appropriate kit according to the molecular weight of the protein to be labeled. The kit provides a 50 KD Filtration tube.
   
2. NHS-Biotin is susceptible to moisture hydrolysis failure, and should be stored at -20°C or -80°C with the desiccant. In order to prevent water vapor from condensing into the NHS-Biotin, it is necessary to equilibrate the NHS-Biotin to room before the experiment.
3. The kit can also be used to label other proteins containing free amino groups. The specific labeling ratio is determined according to the number of available amino groups in the marker or set different molar ratios for labeling.
4. The optimal molecular ratio of the NHS-Biotin and protein (150KD) recommended for labeling with this kit is 20:1, which can ensure that the NHS-Biotin can label the protein, but it cannot ensure the best experimental results. The optimal labeling ratio may vary according to the difference of protein, and the user can optimize according to the actual situation.