Ordering Information
- : 605204-1ML
- : 1 ML
Thrombin, Immobilized, Human Plasma
Description:
Thrombin, Immobilized, Human Plasma
Trade Name:
Calbiochem (EMD Millipore)
Qty/Pk:
1 ML
Background Information:
Native thrombin from human plasma, bound to agarose beads.
Application Notes:
Guidelines for Fusion Protein Cleavage:
Adapted from Xiang, et al (see references)
Please note that this protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature.
1. Immobilized thrombin can be used either with a column or in batch format. For use with batch method the gel may be centrifuged at 3000 rpm or less for separation of agarose from supernatant containing cleaved protein. Higher speeds will result in difficulty resuspending the agarose.
2. A rule of thumb wt/wt ratio to obtain efficient cleavage for most fusion proteins is 1:10 to 1:100 (thrombin:fusion protein)
3. Determine the amount of thrombin required to achieve desired cleavage. Add buffer to this amount of immobilized thrombin to form a slurry. Acceptable buffers include Tris-HCl, containing 150 mM NaCl, citrate buffer, or PBS; the optimal pH is 7.4. For column cleavage, wash the column 2–3 times with buffer prior to addition of sample. The protein should also be resuspended in the same buffer prior to cleavage.
4. Incubation: 25°C, generally overnight; time course including 2 h, 5 h, and 18 h is recommended; assess cleavage by SDS-PAGE. It may be necessary to check cleavage at intermittent intervals (e.g. every 30 min.).
5. For column cleavage, protein that migrates through the column may be monitored at 280 nm.
Adapted from Xiang, et al (see references)
Please note that this protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature.
1. Immobilized thrombin can be used either with a column or in batch format. For use with batch method the gel may be centrifuged at 3000 rpm or less for separation of agarose from supernatant containing cleaved protein. Higher speeds will result in difficulty resuspending the agarose.
2. A rule of thumb wt/wt ratio to obtain efficient cleavage for most fusion proteins is 1:10 to 1:100 (thrombin:fusion protein)
3. Determine the amount of thrombin required to achieve desired cleavage. Add buffer to this amount of immobilized thrombin to form a slurry. Acceptable buffers include Tris-HCl, containing 150 mM NaCl, citrate buffer, or PBS; the optimal pH is 7.4. For column cleavage, wash the column 2–3 times with buffer prior to addition of sample. The protein should also be resuspended in the same buffer prior to cleavage.
4. Incubation: 25°C, generally overnight; time course including 2 h, 5 h, and 18 h is recommended; assess cleavage by SDS-PAGE. It may be necessary to check cleavage at intermittent intervals (e.g. every 30 min.).
5. For column cleavage, protein that migrates through the column may be monitored at 280 nm.
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Brand Family:
Calbiochem
Presentation:
Suspension. In 100 mM NaCl, 20 mM Tris-HCl, ≤0.1% sodium azide, pH 7.4.
Storage Conditions:
Shipped on Blue Ice. Store at +2 - 8°C. Do not freeze.
Product Name:
Thrombin, Immobilized, Human Plasma