Assays for rab5 activity

1. A method of determining intracellular Rab5 activity in a cell sample, comprising the steps of:

providing a fusion protein, wherein the fusion protein comprises a Rab5-binding domain that specifically binds to an activated form of Rab5-GTP, and a tag conjugated thereto, wherein the tag is capable of binding to an affinity matrix for purification of Rab5-GTP;

contacting the fusion protein with an affinity matrix or resin containing a molecule to which the tag of the fusion protein specifically binds;

contacting a cell lysate with the fusion protein bound to the affinity matrix or resin such that Rab5-GTP present in the cell lysate will specifically bind to the fusion protein, thus forming a complex;

rinsing the complex to remove the cell lysate; and

determining the amount of Rab5-GTP present in the complex.

2. The method of claim 1 , wherein the step of determining the amount of Rab5-GTP present in the complex is further defined as subjecting the complex to gel electrophoresis followed by immunoblot analysis with an anti-Rab5 antibody.

3. The method of claim 1 , further comprising the step of determining an amount of total Rab5 present in the cell sample.

4. The method of claim 1 wherein, in the step of providing a fusion protein, the Rab5-binding domain comprises at least a portion of a Rab5 effector protein selected from the group consisting of Rabaptin-5, EEA1(early endosome autoantigen), APPL1 and APPL2 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif 1 or 2), and Rabenosyn-5.

5. The method of claim 1 wherein, in the step of providing a fusion protein, the Rab5-binding domain comprises at least one of SEQ ID NOS:1-3.

6. The method of claim 1 wherein, in the step of providing a fusion protein, the tag comprises at least one of SEQ ID NOS:4-6.

7. The method of claim 1 wherein, in the step of contacting the fusion protein with a matrix or resin, the matrix or resin is selected from the group consisting of glutathione, nickel, zinc, amylase, agarose beads, and combinations thereof.

8. A method of determining intracellular Rab5 activity in a cell sample, comprising the steps of:

providing a fusion protein, wherein the fusion protein comprises a Rab5-binding domain that specifically binds to an activated form of Rab5-GTP, and a tag conjugated thereto, wherein the tag is capable of binding to an affinity matrix for purification of Rab5-GTP;

contacting the fusion protein with an affinity matrix or resin containing a molecule to which the tag of the fusion protein specifically binds;

immobilizing the affinity matrix or resin having the fusion protein bound thereto on a solid support;

contacting a cell lysate with the immobilized fusion protein such that Rab5-GTP present in the cell lysate will specifically bind to the immobilized fusion protein;

eluting the Rab5-GTP from the solid support with an elution buffer; and

determining the amount of Rab5-GTP present in the in the collected elution buffer.

9. The method of claim 8 , wherein the step of determining the amount of Rab5-GTP present in the collected elution buffer is further defined as subjecting the mixture to gel electrophoresis followed by immunoblot analysis with an anti-Rab5 antibody.

10. The method of claim 8 , further comprising the step of determining an amount of total Rab5 present in the cell sample.

11. The method of claim 8 wherein, in the step of providing a fusion protein, the Rab5-binding domain comprises at least a portion of a Rab5 effector protein selected from the group consisting of Rabaptin-5, EEA1 (early endosome autoantigen), APPL1 and APPL2 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif 1 or 2), and Rabenosyn-5.

12. The method of claim 8 wherein, in the step of providing a fusion protein, the Rab5-binding domain comprises at least one of SEQ ID NOS:1-3.

13. The method of claim 8 wherein, in the step of providing a fusion protein, the tag comprises at least one of SEQ ID NOS:4-6.

14. The method of claim 8 wherein, in the step of providing a fusion protein, the affinity matrix is selected from the group consisting of glutathione, nickel, zinc, amylase, agarose beads, and combinations thereof.