Results of 5 μg human IgG1 digestion separated under different quantity of SpeB, The reaction was incubated in PBS with 5 mM DTT, pH 7.4 at 37°C for 1 hour.
M Marker
Lane 1 Negative Control(only UA070131)
Lane 2 Negative Control(only human IgG1)
Lane 3 UA070131-SpeB 2.5 U
Lane 4 UA070131-SpeB 5 U
Lane 5 UA070131-SpeB 10 U
Spe B
Spe B
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Product Details
Product Details
Product Specification
| Species | Streptococcus pyogenes |
| Synonyms | Streptopain, Exotoxin type B, Group A streptococcal cysteine protease (Streptococcal cysteine proteinase), SPE B, Streptococcus peptidase A (SPP) |
| Expression System | E.coli |
| Molecular Weight | 29kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE&HPLC |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 20mM Tris-HCl, 0.5M NaCl, 50% Glycerol (pH 7.0) |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | [1] Musser J M , Stockbauer K , Kapur V ,et al.Substitution of cysteine 192 in a highly conserved Streptococcus pyogenes extracellular cysteine protease (interleukin 1beta convertase) alters proteolytic activity and ablates zymogen processing.[J].Infection & Immunity, 1996, 64(6):1913. |
Background
SpeB is a unique recombinant cysteine protease that can cleave the hinge regions of various species and subclasses of antibodies, thereby generating a homogeneous pool of Fab fragments. This enzyme requires reducing conditions when acting on IgG. SpeB can digest human, mouse, rat, goat and sheep IgG, thereby producing Fab and Fc fragments. The main digestion site of human IgG1 is KTHT/CPPCPAPE.
Components
UA070131: 40U/µL SpeB in 20mM Tris-HCl, 0.5M NaCl, 50% Glycerol (pH 7.0)
5*Reaction Buffer: 5*PBS, 25mM DTT (pH 7.0)
Protocol
1.Take up to 10µg of human IgG1, adjust to 39µl with purified water;
2.Add 10µl of 5*Reaction Buffer;
3.Add 1µl SpeB to a total volume of 50µl, mix gently;
4. React for 1 hour at 37°C.
Guidelines
1. For different antibodies sample, the optimal enzyme concentration and reaction time need to be mapped experimentally;
2. Higher enzyme concentrations increase the digestion efficiency of individual antibodies and need to be optimized accordingly.
Unit Definition
One unit digests ≥ 95% of 1 µg human IgG1 when incubated in PBS with 5 mM DTT or TCEP, pH 7.4 at 37°C for 1 hour.
Picture
Picture
Bioactivity
SDS-PAGE
2μg (R: reducing condition, N: non-reducing condition).
SEC-HPLC
97.9%
RP-HPLC
98%
