Recombinant Staphylococcus Aureus Alpha-Hemolysin (HLY)
Recombinant Staphylococcus Aureus Alpha-Hemolysin (HLY)
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Product Overview
| Description | Recombinant Staphylococcus Aureus Alpha-Hemolysin (HLY) is produced by our E.coli expression system. This is a full length protein. |
| Purity | Greater than 90% as determined by SDS-PAGE. |
| Uniprotkb | P09616 |
| Target Symbol | HLY |
| Species | Staphylococcus aureus |
| Expression System | E.coli |
| Tag | Tag-Free |
| Target Protein Sequence | ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN |
| Expression Range | 27-319aa |
| Protein Length | Full Length of Mature Protein |
| Mol. Weight | 33.4 kDa |
| Research Area | Microbiology |
| Form | Liquid or Lyophilized powder |
| Buffer | Liquid form: default storage buffer is Tris/PBS-based buffer, 5%-50% glycerol. Lyophilized powder form: the buffer before lyophilization is Tris/PBS-based buffer, 6% Trehalose, pH 8.0. |
| Reconstitution | Briefly centrifuged the vial prior to opening to bring the contents to the bottom. Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. It is recommended to add 5-50% of glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C. The default final concentration of glycerol is 50%. |
| Storage | 1. Store at -20°C/-80°C upon receipt, aliquoting is necessary for mutiple use. 2. Avoid repeated freeze-thaw cycles. 3. Store working aliquots at 4°C for up to one week. 4. In general, protein in liquid form is stable for up to 6 months at -20°C/-80°C. Protein in lyophilized powder form is stable for up to 12 months at -20°C/-80°C. |
| Notes | Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week. |
Activity Description – Hemolytic Assay
Assay Type: In vitro hemolytic assay using rabbit red blood cells (RBCs)
Purpose: To confirm the functional activity of recombinant Staphylococcus aureus alpha-hemolysin (HLY) by evaluating its ability to lyse red blood cells, resulting in the release of hemoglobin detected at 540 nm.
Protocol Summary:
1. Protein Reconstitution:
- Lyophilized BLC-00344P was reconstituted in PBS (220 µL protein + 240 µL PBS). The protein dissolved instantly.
2. Controls and Dilution Setup:
Seven tubes were prepared:
- Five tubes with 100 µL PBS (for dilution series)
- One tube with 100 µL deionized water (positive lysis control)
- One tube with 100 µL PBS only (negative control)
3. RBC Preparation:
- 500 µL of 4% rabbit RBC suspension was washed twice in PBS (centrifugation at 2000 rpm, 3 min).
- After final wash, resuspended in 1 mL PBS and gently mixed.
4. Reaction Setup:
- 100 µL of the prepared RBCs were added to each reaction tube.
- Tube 1 showed immediate visible hemolysis.
5. Incubation: Tubes were incubated at 37 °C for 1 hour.
6. Post-Incubation:
- Samples were centrifuged at 2000 rpm for 3 min.
- Supernatants were collected and transferred to a 96-well plate.
7. Detection:
- Absorbance was measured at 540 nm to quantify released hemoglobin.
- Hemolytic activity was confirmed by dose-dependent increases in absorbance.
Result:
The recombinant alpha-hemolysin demonstrated functional hemolytic activity, as indicated by dose-dependent lysis of rabbit red blood cells, confirming preservation of its biological function post-expression and purification.
Target Details
| Target Function | Alpha-toxin binds to the membrane of eukaryotic cells (particularly red blood cells, RBC) forming pores, resulting in hemolysis, with the release of low-molecular weight molecules leading to eventual osmotic RBC lysis. Human RBCs bind much less alpha-toxin than do rabbit RBCs. Heptamer oligomerization and pore formation is required for lytic activity. |
| Subcellular Location | Secreted. |
| Protein Families | Aerolysin family |
