Biotin-HPDP(WS) is a novel water-soluble biotin-labeling reagent that is modified from Biotin-HPDP (Product code: B573). Biotin-HPDP is a conventional reagent to introduce a biotin moiety to a sulfhydryl group of protein via reversible disulfide bond. This reagent is useful for affinity-purification of biotinylated proteins by using avidin-coated beads, because the disulfide bond is cleavable by a reducing agent (Fig.1). Thus, Biotin-HPDP has been widely used for biotin switch assay, which is an analytical technique for protein thiol modifications such as s-nitrosylation, s-sulfhydration, and s-palmitoylation. However, preparation of the solution is time-consuming due to the low-solubility in water, even DMSO and DMF. Therefore, Biotin-HPDP(WS) solution was developed to improve the solubility of Biotin-HPDP.
-SulfoBiotics- Biotin-HPDP(WS) solution is an easy-to-use aqueous solution including 20mmol/l Biotin-HPDP(WS).
Figure 1 Schematic Protocol for Biotin Labeling with Biotin-HPDP(WS) and Purifiation of the Biotinylated Protein
– Biotin Labeling to GAPDH and the Recovery with Avidin Beads –
< Procedure >
1. Lysis buffer (35 μl) and 100 mmol/l DTT in Lysis buffer (5μl) were added to 10 μl of 1 mg/ml GAPDH solution in PBS in a 1.5 ml-microtube, and mixed by vortex.
2. After the solution was incubated at 37 ℃ for 30 minutes, the whole solution was transferred to a 10 K filtration tube, and centrifuged at 12,000 rpm for 10 minutes.
3. PBS (50 μl) was added to the filtration tube, and centrifuged at 12,000 rpm for 10 minutes.
4. Step 3 was repeated.
5. RIPA buffer (126 μl) and 4 mmol/l Biotin-HPDP(WS) solution in H2O (14 μl) were added to the filtration tube, and mixed with the protein by pipetting.
6. The filtration tube was incubated at 37 ℃ for 1 hour, and centrifuged at 12,000 rpm for 10 minutes.
7. PBS (50 μl) was added to the tube, and centrifuged at 12,000 rpm for 10 minutes.
8. Step 7 was repeated.
9. Neutralization buffer (400 μl) was added to the tube to dissolve the biotin-labeled protein by pipetting, and the solution was transferred to a 1.5 ml-microtube.
10. The solution (50 μl) of Step 9 was added to NeutravidinTM Agarose beads in a tube. ※ Neutravidin Agarose beads were washed with Neutralization buffer prior to the reaction.
11. The solution was incubated at 4 ℃ for 1 hour.
12. The tube was centrifuged at 2,500 rpm for 1 minute, and the supernatant was removed using a pipette.
13. Neutralization buffer (+600 mmol/l NaCl) (1 ml) was added to the tube and centrifuged at 2,500 rpm for 1 minute, andthe supernatant was removed using a pipette.
14. Step 13 was repeated twice.
15. Neutralization buffer (1 ml) was added to the tube and centrifuged at 2,500 rpm for 1 minute, and the supernatant was removed using a pipette.
16. Step 15 was repeated.
17. Elution buffer (50 μl) was added to the tube, and mixed by vortex. The solution was incubated at 4 ℃ for 1 hour.
18. The tube was centrifuged at 2,500 rpm for 1 minute, and 10 μl of the supenatant was transferred to a 1.5 ml-microtube.
19. Loading buffer (2 μl) was added to the microtube, and the solution was applied to SDS-PAGE (CBB staining) and western blotting.
Figure 2 Detection of recovered GAPDH
Comparison between Biotin-HPDP and Biotin-HPDP (WS)
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