The overall goal of this procedure is to use a method known as Tandem affinity purification to isolate protein binding partners. Hello, I'm dLAN Bailey from the section of Virology at Imperial College London. In our lab, we work on virus host interactions for different members of the virus family.
Identifying proteins that interact with other cellular or viral proteins is a great starting point for investigating the interplay between the virus and its host during infection. In this video, I'll explain one approach that we use in our lab, tandem and e purification or tap tagging. The Tap tag used in this study is an N terminal tag consisting of two units of protein G and a strep adin binding peptide.
These are separated by A TEV protease cleavage sequence that allows specific elution the bait protein. In this example, murine, EIF four E is at the C terminus of this fusion construct. The tap tagging protocol is a six step procedure.
After transfection and generation of cell lines, cells are induced to express the tag abate protein before being lies in a mild lysis buffer. Two affinity purification steps and two specific solutions are used to purify the bait and any potential interacting partners to generate cell lines. HEC 2 93 cells should be transfected with the P met four expression vector encoding.
The tap tagged bait protein cells are then selected with a hundred micrograms per mil hygromycin B.Since the plasmid is maintained episo, there is no need to isolate specific clones. 10 confluent flass of cells are induced by treatment with 10 micromolar cadium chloride for 16 hours before being scraped off into media. The suspended cells are then transferred to 15 M tubes and the cells pelleted by centrifugation.
These cells are then washed three times with ice cold PBS before finally being pelleted. Step two cell lysis cells are lies in five mils of cold lysis buffer by repeated pipetting before being left on ice for five minutes. To ensure efficient lysis, the cells are then syringed through a narrow gauge, blunt needle and freeze thaw.
The resultant sate is allotted into 1.5 mil tubes and unli cells and debris removed by centrifugation. Of note, the pellet size is markedly reduced following this centrifugation step. The lysate supernatants are then combined in a 15 mil Falcon tube and passed through a 0.45 micrometer filter to remove any additional debris.
A 50 microliter aliquots of this lysate should be taken for subsequent analysis. This sample is referred to as sample one. Step three, binding to rabbit IgG Aeros.
In this stage of the protocol, the protein G tags are immuno precipitated by rabbit IgG aros. To allocate aros beads, remove the end of ape tip. This helps to avoid damaging the beads.
Gently resuspend the rabbit IgG aro solution by swirling the bottle before removing the aros to a 15 mil tube. Wash the aros three times in chilled lysis buffer using low speed centrifugation to clarify the beads at each step. The pelleted rabbit IgG beads should be visible after centrifugation.
After each step, carefully remove the buffer without disturbing the beads. After the final wash is complete, add the filtered and clarified lysate to the packed beads and incubate for three hours or overnight. It preferred at four degrees C using a rotating mixer.
Following the immunoprecipitation, spin down the agros beads and remove the supernatant for subsequent analysis. If required, you can use a fine build tip to remove any remaining liquid from this tube. This supernatant is sample two step four TEV protease cleavage.
In this step, the bait is cleaved from the protein G tags. This step allows specific cleavage of the bait protein effectively an elution step from the rabbits IgG aros immunoprecipitation. Prepare the TEV cleavage mix and add this to the aros beads using ape tip with its end removed, transfer this mixture to a siliconized micro centrifuge tube and invert to mix the aros into suspension prior to overnight incubation at four degrees C, remove an alico of this TEV cleavage reaction for analysis.
This is sample three. Overnight incubation on the rotating mixer should allow the TEV reaction to progress to near completion. Step five, binding to immobilize strept adin beads.
In this step, the Cleve bait and any potential binding partners are affinity purified from solution. Firstly, centrifuge the TEV cleavage reaction containing the rabbit IgG aros beads. To pellet the aros, remove an OTT of the clarified supernatant for analysis.
This is sample four. Carefully remove all the remaining supernatant, which contains your bait protein to a fresh tube. Again, use fine.
Build peppe tips to remove all possible supernatant. Retain the rabbit IgG aros beads for subsequent analysis. This is sample five.
To prevent damage to the strep adin beads, remove the end from a pet tip. Gently resuspend the beads and remove an alico to a siliconized tube. Wash the strep having in beads with S buffer, clarifying by low speed centrifugation.
After each wash. After each wash, carefully remove the buffer. Strept adin beads are very small and can be easily displaced even when using fine.
Build pet tips. After the strept adin beads have been washed. Add the supernatant from the TEV cleavage reaction to these beads and incubate the reaction for three hours or overnight at four degrees C, using a rotating mixer.
After incubation centrifuge has strept havein beads and remove the sup natum. This is sample six. Of note.
The Beit protein is now associated with the remaining beads in the tube. Wash the streptavidin beads containing your bait protein with lysis buffer. To remove any additional contaminating proteins, remove the remaining wash solution from the beads and leave them on ice.
Step six, biotin elution. In this step, biotin is added to the streptavidin beads. The interaction between biotin and the streptavidin beads displaces the bait, which goes back into solution.
Add the biotin solution directly to the beads and invert to mix. Incubate the reaction at four degrees C for three hours or overnight using a rotating mixer centrifuge, the biotin aeros mixture and carefully collect the mixture into a fresh tube. This is your final EIT referred to as sample seven.
The remaining strept havein beads are sample eight. Because of its low protein concentration, this final eluate needs to be concentrated prior to analysis. This is achieved using low molecular weights, cut off spin columns, reduce the volume by centrifugation monitoring the process regularly.
This concentrated sample can be split and analyzed on SDS page gels by kumasi and silver stain. If the samples are to be analyzed by mass spec, we advise using precast commercial gels. Individual bands can then be extracted and the proteins identified by mass spec.
In this case, the bait was murine, EIF for E protein. The advantage of the tap system is you have the ability to express your protein of interest in a particular cell line, and depending on the cell line that you are interested in, it also gives you the ability to express the protein at relatively low levels. You decide to, and all post-translational modifications and localization retained, so you can often purify the native complex.
