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Proteomics & Protien Expression
Proteins from Inclusion Bodies
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ABSTRACT
The expression of foreign proteins at high levels in E. coli often results in the formation of inclusion bodies composed of insoluble aggregates of the expressed protein. The inclusion bodies are recovered from bacterial lysates by centrifugation and are washed with Triton X-100 and EDTA to remove as much bacterial protein as possible from the aggregated foreign protein.
To obtain soluble protein, the washed inclusion bodies are dissolved in denaturing agents and the released protein is then refolded by gradual removal of the denaturing reagents by dilution or dialysis. Whereas the procedure given here has been used to solubilize inclusion bodies, each protein may require a slightly different procedure, which must be determined empirically. |
| Products Available for this Protocol |
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Buffers, Solutions, and Reagents |
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| Tris-Cl (pH 8.0) |
T5941 |
Trizma® hydrochloride |
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| NaCl |
S3014 |
Sodium chloride |
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| EDTA |
E7889 |
Ethylenediaminetetraacetic acid disodium salt solution |
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| Triton X-100 |
T8787 |
Triton® X-100, for molecular biology |
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| Deoxycholic acid, protein grade |
D4297 |
Deoxycholic acid |
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| HCl |
H1758 |
Hydrochloric acid |
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| Urea |
U5378 |
Urea |
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| Phenylmethylsulfonyl fluoride (PMSF), fresh |
P7626 |
Phenylmethanesulfonyl fluoride |
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| KH2PO4 |
60353 |
Potassium phosphate dibasic |
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| KOH |
P5958 |
Potassium hydroxide |
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| Isopropanol |
I9516 |
2-Propanol, for molecular biology |
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| Enzymes and Gel Loading Buffers |
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| DNase I |
AMPD1 |
DNase I, Amplication Grade |
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| Lysozyme |
L6751 |
Lysozyme from chicken egg white, lyophilized powder |
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| Sodium dodecyl sulfate (SDS) |
L3771 |
Sodium dodecyl sulfate |
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| Glycerol |
G5516 |
Glycerol for molecular biology |
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| Dithiothreitol (DTT) |
D9779 |
DL-Dithiothreitol for molecular biology |
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| Bromophenol blue |
B5525 |
Bromophenol Blue sodium salt |
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| Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest. |
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