How to remove Factor Xa?

After protease digestion, Factor Xa Protease can be removed by affinity chromatography using Xa Removal Resin. The Xa Removal Resin binds the protease in the reaction mixture while the cleaved recombinant protein remains in solution.

What does factor Xa cleave?

Factor Xa is a serine protease which cleaves prothrombin to generate thrombin and lies at the crossroads of the extrinsic and intrinsic coagulation pathway.

What is factor Xa site?

Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-Glu/Asp-Gly-Arg. It will sometimes cleave at other basic residues, depending on the conformation of the protein substrate. The most common secondary site, among those that have been sequenced, is Gly-Arg.

How do you get rid of thrombin?

Non-tagged proteases such as thrombin or Factor Xa can be removed using a Benzamidine resin, which binds to serine proteases. Tagged proteases can be removed with the same affinity chromatography resin/column used to capture the tagged protein of interest.

What is the function of factor Xa?

Coagulation factor Xa is a protein that reverses the effects of certain anticoagulant medications that are used to treat or prevent blood clots. Reversing anticoagulant medicine is necessary if you have uncontrolled or life-threatening bleeding as a result of how that medicine works.

What does a high anti Xa level mean?

A high level of anti-Xa may be seen if the patient has renal impairment (in the case of LMWH)or if the specimen is contaminated with heparin (specimen drawn from lines containing heparin).

What is factor Xa called?

Factor Xa is the activated form of the coagulation factor X, known eponymously as Stuart-Prower factor. Factor X is an enzyme, a serine endopeptidase, which plays a key role at several stages of the coagulation system.

What is factor Xa used for?

Coagulation factor Xa is used to treat uncontrolled bleeding in people who take the anticoagulants rivaroxaban (Xarelto) and apixaban (Eliquis). Coagulation factor Xa was approved by the US Food and Drug Administration (FDA) on an “accelerated” basis.

How do I cut off his tag?

The easiest way is to use TEV protease (very specific) which also has a HIS tag itself. After cleavage (30C 4h or 4C overnight) you can run another Ni-NTA column, but this time, collect the flow-through, because the column will bind the cleaved tag and TEV protease itself. For us it works perfectly fine.

What is a normal anti-Xa level?

Anti-factor Xa Anti-Xa levels should be checked at their peak at 4 hours after dosing (both q12 and q24 variations). Reference ranges are not clinically validated and can vary by facility and indication for use. Suggested “therapeutic range” is usually 0.6-1.0 units/mL.

How is the factor Xa assay kit developed?

Factor Xa Assay Kit (ab154128) is developed to determine Factor Xa activity in human plasma, serum and cell culture samples. The assay couples immunofunctional and direct amidolytic function. A polyclonal antibody specific for human Factor Xa has been pre-coated onto a microplate and Factor Xa is bound to the immobilized antibody.

How is factor Xa protease removed after digestion?

After protease digestion, Factor Xa Protease can be removed by affinity chromatography using Xa Removal Resin. The Xa Removal Resin binds the protease in the reaction mixture while the cleaved recombinant protein remains in solution. After the resin is pelleted by centrifugation, the cleaved recombinant protein is recovered in the supernatant.

How much protein can be cleaved by Factor Xa?

Concentration of protein to be cleaved:Factor Xa Protease activity is sensitive to the concentration of protein to be cleaved. A minimum of 10 µg protein per 40 µl reaction (0.25 µg/µl) is recommended. Incubation temperature:Factor Xa Protease activity increases with increasing incubation temperature from 4°C to 37°C.

How to check anti factor Xa level before use?

Anti Factor Xa reagent:Invert to mix before use. Calibrators:If required, reconstitute one vial of each concentration (0, 0.8, 2.0 IU/mL) with 1.0 mL of CLSI Type CLRW water or equivalent. Replace the stopper and keep controls at 15-25ºC for 30 minutes. Ensure the complete reconstitution of the calibrators.