This protocol uses the interaction between poliovirus and Poliovirus, recognizing nano bodies to demonstrate a simple quantitative liquid phase affinity capture assay. Principally cobalt coated magnetic beads are used to pull down tagged proteins as well as any interacting radio labeled proteins. First, mix the labeled and tagged proteins and incubate at room temperature.
Then add magnetic beads that recognize the tag, separate the bound fraction of labeled protein from the unbound fraction using magnets. Then measure the signal of the labeled protein found in the unbound fraction and determine the amount of labeled protein found in the unbound fraction. Results from this liquid phase affinity assay are extremely useful when confirmational conversion sensitive proteins are assayed.
This liquid phase affinity capture assay can provide insight into the interaction between poliovirus, recognizing nano boies and different poliovirus antigens. The method can also be applied to other protein protein interactions provided that one protein can be labeled and the other one can be detected. We first had the ID for this methods when we were interested in investigating the interaction between poliovirus, recognizing nano bodies and different poliovirus antigens.
Since poliovirus is sensitive to confirmational conversion, when attached to a solid surface, the use of e LSA is limited. A liquid phase based system should therefore be preferred. An example of such a liquid phase based system often used in poliovirus research is the micro protein.
A immunoprecipitation assay. Even though this test has proven its applicability, it requires the crystallized structure of a conventional antibody, which is absent in nano bodies. These nano bodies, which are interesting and very stable single domain antibodies, can be easily engineered with different text.
The widely used. He stack shows affinity for bivalent ions, such as nickel and cobalt, which on their turn can be easily coated on magnetic beads. We therefore developed the simple quantitative affinity capture assay based on cobalt coated magnetic beads.
Reese's the stock of magnetic beads by vortex and transfer 10 microliters for each sample into a tube pellet the beads using a magnet. Remove the clear supinate to wash the beads. Add 150 microliters of binding buffer.
Harvest the beads using a magnet and remove the supinate after a second wash. Re suspend the beads in 40 microliters of binding buffer for each sample to control. For the background in sample one, prepare a well without radiolabeled virus, but equal volume of binding buffer for control.
Sample two of 100%radio activity. Add all components into a well except substitute the nano body with binding buffer. Now add 2000 counts per minute of S 35 labeled poliovirus seven strain type one in a total volume of 80 microliters of binding buffer to all samples.
Accept control. Sample one. Add 10 microliters of nano body dilution to each sample.
Well mix for 10 seconds on a shaker and incubate for one hour of room temperature. Next, add 40 microliters of the washed magnetic bead suspension and incubate for 10 minutes at room temperature under continuous shaking, pellet the beads with a magnet and transfer the cleared SUP natant into a tube to measure radioactivity of each sample, transfer 50 microliters of the supinate into a counting flask. Add three milliliters of scintillation fluid mix and read on this insulation.
Counter pull the used magnetic beads in a tube using water and centrifuge 500 times G for two minutes. Discard the supinate and re suspend the beads in six milliliters of 0.5 molar sodium hydroxide. Then aliquot the suspension into multiple tubes and incubate in an ultrasonic bath for five minutes.
After pelleting the beads with a magnet and removing the supinate, add one milliliter of 2%SDS solution to each tube, then mixed by vortexing and foil for five minutes. Now, harvest the beads. Add one milliliter of 0.2 molar EDTA pH seven to each tube, and incubate the tubes in an ultrasonic bath for five minutes.
Then collect the beads, wash beads with one milliliter of water three times followed by one milliliter of 10 millimolar cobalt chloride solution. Place tubes on a shaker for 10 minutes. Proceed to harvest beads on a magnet and resus suspend in one milliliter of binding buffer.
Wash twice in one milliliter of 20%Ethanol finally resuspend the beads in their original volume of 20%ethanol to obtain regenerated beads in a concentration of 40 milligrams per milliliter. Correct for background radioactivity by setting control. Sample one as the 0%radioactivity value and control sample two as the 100%radioactivity value.
Now for each sample, calculate the percentage of radioactivity in the sate as a measure of the overall precipitation of the radio labeled virus by the nano body. The this experiment evaluates the affinity of PVSS 38 C and antibody specific for poliovirus using three different antigens of poliovirus, native antigen, heated antigen, and 14 s subunits. Interestingly, radioactivity in the sate of the samples containing radio labeled 14 s subunits decreases with increase in concentrations of the nano body PVSS 38 C.This indicates an increase in the amount of Poliovirus 14 s subunits connected to the nano body P pvs, S 38 C, and indirectly to the magnetic beads.
Since no affinity of pvs S 38 C for the N antigenic and H antigenic form of poliovirus is observed, the nano body appears specific for the 14 s subunit of poliovirus. The affinity of the nano body towards its antigens as measured by this method is specific as the unrelated nano body. NB one shows no reactivity against the three antigenic forms of poliovirus.
This system produces high reproducibility as indicated in this dose response curve generated by different investigators on different days. Once mastered this reliable liquid phase, affinity capture assay can be properly performed within two hours recycling of the beats included. After watching this video, you should have a good understanding of how to use this liquid phase affinity capture assay based on magnetic beats when studying the interaction between different proteins, especially when solid phase based systems cannot be used.
