Description
| Product Name: | Biotin Labeling Kit |
| SKU: | AKES126 |
| Labeling group: | NHS-Biotin |
| Applications: | Protein labeling/Antibody labeling (50 KD Filtration tube) |
Biotin Labeling Kit provides all the reagents required for labeling, which can label antibodies and other proteins containing primary amino-group (-NH2) molecules simply and effectively.
| Products | 1 Reaction | 3 Reactions | 10 Reactions | Storage |
| AF 568 NHS ester | 0.12 mg×1 | 0.12 mg×3 | 0.12 mg×10 | 20°C, shading light |
| Labeling Buffer II | 10 mL | 20 mL | 20 mL x 2 | 2-8°C |
| DMF | 500 uL | 500 uL | 500 uL | 2-8°C, shading light |
| 1×PBS(pH7.4) | 10 mL | 10 mL | 10 mL x 2 | 2-8°C |
| 1M Tris(pH 8.7) | 500 uL | 500 uL | 500 uL x 2 | 2-8°C |
| 3 KD Filtration tube* | 1 set** | 3 sets | 10 sets | RT |
Material Required
- Pipettor and tips (0.5-10μL, 2-20μL, 20-200μL, 200-1000μL).
- 37°C incubator.
- Centrifuge (centrifugal force up to 12,000×g).
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.
| 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 5 mL, cover the filter tube, centrifuge at 12,000×g for 5 min, and discard the liquid in the collection tube. |
| Note: | The maximum volume of the filter device is 5 mLIf the volume of 1 mg protein is greater than 5 mL, please add it in several times and concentrate it by centrifugation and ultrafiltrationIf 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 full. |
| 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 5 mL Labeling Buffer II into the filter device and put it on a pipe rack for later use. |
| Labeling reaction: | Immediately add 3 μL of 10 mM AF 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. |
| Blocking: | Add 1 M Tris (pH 7) to stop the reaction at the ratio of 10 μL of 1 M Tris (pH 7) per 100 μg protein, mix fully and incubate at room temperature for 10 min. |
| Ultrafiltration & purification: | Add an appropriate amount of 1×PBS into the above reaction solution to the final volume of 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 5 mL, it can be transferred to the spin-dried filter device for several times after ultrafiltratio, and cover the 5 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. |
| Collect labeled product: | Add 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. |
| Storage: | 2~8°C/-20°C, shading light |
| Expiration date: | 12 months |
| Shipping: | Ice bag |
