Western blot by incorporating an affinity purification zone
A mixture of components is flowed through a binding channel region comprising a component-binding moiety, thereby binding at least a portion of a component of interest. The mixture is then flowed through a separation channel region that includes a buffer comprising a detergent, resulting in separated components. Diluent is mixed with the separated components, diluting the detergent, and the separated components are detected. The component of interest is released from the component-binding moiety and flowed through the separation channel region. Diluent is mixed with the released component of interest, diluting the detergent, and the released component of interest is detected.
1. A method of detecting a component of interest in a mixture of components, the method comprising:
(i) flowing the mixture of components through a binding channel region, which binding channel region comprises a component-binding moiety, thereby binding at least a portion of the component of interest to the component-binding moiety;
(ii) flowing the mixture of components through a separation channel region, resulting in one or more separated components, wherein the separation channel region comprises a separation buffer, which buffer comprises a detergent;
(iii) mixing a first portion of diluent with the one or more separated components, thereby diluting the detergent;
(iv) detecting the one or more separated components;
(v) releasing the component of interest from the component-binding moiety, resulting in a released component of interest;
(vi) flowing the released component of interest through the separation channel region;
(vii) mixing a second portion of diluent with the released component of interest, thereby diluting the detergent; and
(viii) detecting the released component of interest.
2. The method of claim 1 , wherein the separation buffer further comprises a dye moiety, and wherein detecting the one or more separated components or the released component of interest comprises detecting the dye moiety, which dye moiety associates with the one or more separated components or the released component of interest.
3. The method of claim 2 , wherein the dye moiety comprises a lipophilic dye.
4. The method of claim 1 , wherein diluting the detergent comprises diluting the detergent to a level below a critical micelle concentration.
5. The method of claim 1 , wherein the component of interest comprises a protein and the component-binding moiety comprises a protein-binding moiety.
6. The method of claim 1 , wherein the component-binding moiety comprises an antibody specific to the component of interest.
7. The method of claim 1 , wherein the component of interest comprises a carbohydrate and the component-binding moiety comprises a carbohydrate-binding moiety.
8. The method of claim 1 , wherein the carbohydrate-binding moiety comprises a lectin specific to the carbohydrate.
9. The method of claim 1 , wherein the component-binding moiety or the component of interest comprises a label moiety.
10. The method of claim 1 , wherein the binding channel region comprises a derivatized channel.
11. The method of claim 1 , wherein the binding channel region comprises a particle set, the particle set comprising a plurality of particle member types.
12. The method of claim 11 , wherein binding the component of interest to the component-binding moiety comprises adsorbing the component of interest onto one or more members of the particle set.
13. The method of claim 1 , step (v) comprising releasing the component of interest from the component-binding moiety by adjusting the temperature or pH in the binding channel region or by introducing one or more releasing reagents into the binding channel region.
14. The method of claim 1 , further comprising contacting the mixture of components with a detergent and heating the mixture of components after step (i) and prior to step (ii).
15. The method of claim 1 , further comprising contacting the mixture of components with a detergent and heating the released component of interest after step (v) and prior to step (vi).
16. The method of claim 1 , further comprising determining the molecular weight of the separated components or the released component of interest by determining the retention time of the separated components or the released component of interest in the separation channel region.
17. The method of claim 1 , wherein detecting the one or more separated components produces a first signal and detecting the released component of interest produces a second signal, and wherein the method further comprises deconvoluting the first signal and the second signal to identify the separated components and the component of interest.
18. The method of claim 1 , wherein detecting the one or more separated components produces a first data set and detecting the released component of interest produces a second data set.
19. The method of claim 18 , further comprising superimposing the first data set and the second data set to identify the component of interest.
20. The method of claim 1 , wherein the released component of interest has a migration time through the separation channel region, the method further comprising identifying the component of interest using the migration time.