Ab-10 Rapid Biotin Labeling KitLK37 Product Manual

Ab-10 Rapid Biotin Labeling Kit enables rapid (in less than 30 min) and easy labeling of Biotin to 10 μg antibody.Reactive Biotin (a component of the kit) has a succinimidyl ester group, that can easily make a covalent bond with an amino group of the target antibody without any activation process. This kit contains all the necessary reagents to prepare a Biotin-labeled antibody except for DMSO.
 

Fig. 1 Labeling procedure

Caution After a Reactive Biotin is taken out from the seal bag, keep the unused Reactive Biotin in the bag, seal tightly and store at -20°C. Store the other components at 0-5°C.

Reactive Biotin*	×3
Reaction Buffer	100 μl ×1
Stop Solution	100 μl ×1

• Though it is hard to see the reagent Reactive Biotin because of its small amount, it is attached on the bottom of the tube as a sheet form. Please collect the Reactive Biotin carefully by pipetting with buffer as described in Step 4 of the protocol.

Store at 0-5 °C
This kit is stable for 1 year at 0-5°C before opening.

• 20 μl, 200 μl adjustable pipettes
• Microtube (for sample and Working buffer preparation)
• Incubator (37 °C)
• PBS (Phosphate buffered saline)
• DMSO (Dimethyl sulfoxide)

• Use 0.5-1 mg/ml of antibody solution for labeling. If the antibody concentration is more than 1 mg/ml, please dilute the antibody solution with PBS.
• If the sample solution contains small insoluble materials, centrifuge the solution, and use the supernatant for the labeling.
• The microtubes in this kit contain solutions. Since there is a possibility that the droplets might attach to the inside walls or caps, please spin the tube to drop them down prior to open.
• In case an antibody solution includes a high concentration of constituents, such as BSA or glycerol, it may interfere with a labeling and cause a non-specific signal. We recommend removing the constituents prior to labeling procedure. Usable constituents (○) and non-usable constituents (×) are shown in Table 1, and compatible concentrations of constituents are shown in Table 2.

Table 1. Usable/non-usable constituents

Additivea
Buffering agents (PBS,HEPES)	〇
Sodium chloride	〇
Chelating agents (EDTA)	〇
Sodium azide	〇
Sugars (Glucose, Trehalose)	〇
Primary amines and thiols	×

 

Table 2. Compatible concentrations of constituents

	Glycerol	BSA	Gelatin	Tris
Anti-Chloramphenicol Acetyl Transferase (CAT) antibody	< 20%	< 0.1%	< 0.1%	< 50 mmol/l
Anti-GAPDH antibody	< 50%	< 0.1%	< 0.1%	< 50 mmol/l
Anti-CD44 antibody	< 50%	< 0.5%	< 0.1%	< 50 mmol/l

• Interference and non-specific signal may be dependent on types of antigen, host species of antibody or constituents.

1. Add Reaction Buffer (up to 30 μl) to a microtube and mix it with an equal volume of DMSO to prepare Working buffer.
2. Add 0.5-1 mg/ml of the antibody solution to another microtube to be an amount of antibody of 10 μg.
3. Add Working buffer (step.1) to the antibody solution (step 2) and mix by pipetting.
   • The volume of Working buffer : one-fifth of the antibody solution (Table 3).
4. Add the solution (step 3) to Reactive Biotin and mix by pipetting.
5. Incubate at 37°C for 10 minutes.
6. Add 28 μl of Stop Solution to the solution (step 5) and mix by pipetting.
7. Incubate at 37°C for 10 minutes.
8. Apply the sample (step 7) for desired experiments or store at 0-5°C.
   • The labeled antibody is stable at 4oC for 2 weeks.

Table 3. The volume of Working buffer

The concentration of antibody (mg/ml)	0.5	0.6	0.7	0.8	0.9	1.0
The volume of Working buffer (μl)	4.00	3.34	2.86	2.50	2.22	2.00

 

Mitochondria immunostaining

1. HeLa cells were seeded on a μ-slide 8 well (ibidi) and cultured overnight at 37 °C in a 5% CO₂ incubator.
2. The cells were washed with PBS three times, and 4% paraformaldehyde in PBS was added to the μ-slide.
3. The μ-slide was incubated at room temperature for 15 minutes.
4. The cells were washed with PBS three times, and 1% Triton-X in PBS was added to the μ-slide.
5. The μ-slide was incubated at room temperature for 30 minutes.
6. Once the cells were washed with PBS three times, a blocking solution prepared with PBS was added to the μ-slide.
7. The cells were then incubated at room temperature for 1 hour.
8. Biotin conjugated anti-mitochondria antibody was diluted 50 times with the blocking solution.
   • Anti-mitochondria antibody was purchased from Abcam (Product Code: ab3298) .
9. The supernatant was discarded and the solution (step 8) was added to the μ-slide.
10. The μ-slide was incubated at 0-5oC overnight.
11. The supernatant was discarded and the cells were washed using PBS-T three times.
12. 0.2 μg/ml peroxidase conjugated streptavidin was added to the μ-slide.
13. The μ-slide was incubated at room temperature for 1 hour.
14. The supernatant was discarded and the cells were washed using PBS-T three times.
15. The cells were washed using Tris buffer (TB, 50 mmol/l, pH 7.5) three times.
16. The supernatant was discarded and DAB solution [0.2 mg/ml DAB (Dojindo Laboratories, Product Code:D006),
    0.003% H₂O₂ , 50 mmol/l Tris (pH 7.5)] was added to the μ-slide.
17. The μ-slide was incubated at room temperature for 10 minutes.
18. After the cells were washed using TB three times, TB was added to the μ-slide.
19. The cells were observed under a microscope.

Fig. 2 Microscope image of DAB-stained mitochondria in HeLa cells