Hello, I am Christina McKennan from the University of Helsinki, Finland. One of the challenges in characterizing a particular protein complex is to determine its molecular composition. This requires development of methods to isolate and purify the complex from its cellular environment.
The area of research in microbe is plant virus host interactions and therefore we are interested in the various viral ripon nuclear protein complexes formed during infection. In the past, we have successfully studied these complexes using affinity duct based purification in our hands. Twin strep duct proved to be one of the best ducks.
We have, however, observed certain limitations in its use, one of them being low yield and therefore we wanted to improve this method. Hello, my name is Mata and I will guide you through the experimental protocol. But first, let me give you an overview of the method.
We employ strep tachin macro prep beads to purify specific complexes between twin strept tagged proteins and their physiological binding partners. Strep strept tagged and tetramer is shown in green twin strept tagged protein In blue, its binding partner in orange and two copies of the strep tag. In red.
The unbound proteins depicted as grace fears are removed by washing in the canonical approach. The bound complexes are competitively alluded with biotin shown in pink. However, while using this approach, we observed incomplete elution of bound complexes with only a fraction successfully detaching from the strep tachin beads.
This results in low target protein recovery. Complicating any further analysis, target protein recovery can be improved by using denaturing elution with S-D-S-S-D-S. Miss cells are formed around the denatured twin strep ttag bait protein.
Its binding partner and around the dissociated monomers of strept actin. The resulting sample contamination with released strept actin is unacceptable if the sample needs to be further analyzed by mass spectrometry. To overcome the sample contamination problem, we developed a method whereby strep tachin tetramer is first stabilized by chemical crosslinking.
The resulting crosslinked resin is used to purify the bait protein and its binding partner sample elution with SDS ensures high protein recovery while chemical crosslinking of the resin ensures elution of the strept tagged bait protein and its binding. Partner in the absence of sample contamination with released strept equilibrate one sealed no way. Micro tube containing two milligrams of BS three crosslinker to room temperature.
Prepare a pipette tip for resin transfer by cutting the end of the tip off reus resuspend strep tact and macro prep resin by brief vigorous shaking and immediately transfer 600 microliters of the suspension to a CoStar spinx filter column centrifuge at 4, 000 RPM for 30 seconds. At room temperature add 450 microliters of phosphate buffered saline. The purpose of this step is to replace primary am mean triss with non-reactive phosphate.
Discard the flow through. Mix the contents by flipping the tube several times. Repeat the centrifugation and wash steps two times and leave the resin in 430 microliters of P-B-S-P-H eight.
After the last centrifugation, puncture the foil of the no way micro tube with a pipette tip containing 100 microliters of milli Q.Water dissolve the BS three powder in water by gently pipetting up and Down. Once the BS three is completely dissolved immediately add 20 microliters of the solution to the spin X column. Ensure that the contents are mixed properly.
Rotate the column for 30 minutes at room temperature, making sure that the resin is mixed properly with the BS three solution. To quench the reaction, add six microliters of three molar triss adjusted to pH 7.5 with hydrochloric acid. Rotate the column for another 15 minutes at room temperature centrifuge at 4, 000 RPM for 30 seconds.
At room temperature reus, suspend the cross-linked resin in 450 microliters of tris buffered saline with tween 20 and discard the flow through. Repeat the centrifugation and wash steps two more times At the last step, resuspend the resin in 450 microliters of TBS. Transfer the resin suspension to a fresh tube resuspend the resin left in the column with another 450 microliters of TBS and transfer to the same tube.
Repeat the last step once more to ensure maximum transfer of the resin from the column to the tube. Let the tube with the resin suspension stand for 10 minutes at room temperature and adjust the volume to 600 microliters. By removing excess TBS, the resin is ready for immediate use or can be stored at four degrees Celsius without freezing.
This method is suitable for purification of any twin strep tagged fusion protein and its associated complexes. The protein can be of animal plant or bacterial origin and can be isolated from either total cell lysate or enriched organal fraction. In this example, we isolate a twin strep tag protein VPG Pro of potato virus A from the nuclear fraction of virus infected plants begin by centrifuging one milliliter of the bait protein sample in binding buffer at 13, 000 RPM for 10 minutes at four degrees Celsius.
Transfer the supernatant to a fresh tube to minimize binding of endogenous biotinylated proteins to the strep tact and resin. Add avadon to a final concentration of 100 micrograms per milliliter, making sure that the contents are thoroughly mixed. Rotate for 15 minutes at four degrees Celsius.
Resuspend the cross-linked resin by vortexing and using a cup pipette tip immediately add 50 microliters of the resin suspension to the tube containing the bait protein sample. Rotate for another 30 minutes at four degrees Celsius while waiting. Set the thermo mixer to 55 degrees Celsius and preheat 500 microliters of Elu buffer for use in the elucian step centrifuge at 2000 RPM for one minute at four degrees Celsius.
Discard the supernatant and wash the resin with one milliliter of pre chilled wash buffer. Number one, rotate for five minutes at four degrees Celsius. Repeat the centrifugation and wash steps three times at the last step.
Resuspend the resin in 250 microliters of wash buffer number two and transfer the resin suspension to a fresh spinx column. Resuspend the resin remaining in the tube in another 250 microliters of wash buffer number two and transfer to the same column centrifuge at 2000 RPM for three minutes at four degrees Celsius. Transfer the column to a fresh dolphin nose two milliliter tube and discard the flow through.
Proceed immediately to the following elucian step. Add 150 microliters of preheated elution buffer to the spinx column. Incubate the solution in the thermo mixer for five minutes at 55 degrees Celsius, shaking at 1400 RPM centrifuge at 4, 000 RPM for one minute at room temperature And discard the column.
The purified target proteins are now ready for further analysis and can be stored below minus 20 degrees Celsius is the image demonstrates that protein elution from strept actin macro prep resin with biotin is incomplete compared to elution with SDS. The difference in western blot signal intensities reflect different protein yields obtained with the two elucian techniques. Although elucian with SDS is much more efficient than elucian with biotin, it leads to sample contamination with released strept actin.
The image shows silver stain gel of SDS EITs from the Strept actin Macro prep resin note, the presence of released strept actin in the left lane but its absence in the right lane. The sample on the left was alluded from the non cross-linked resin and the sample on the right from the resin cross-linked with BS three. Based on these results, we conclude that cross-linking with BS three prevents the release of strept actin from the resin during SDS elution.
In this example we used Strept actin macro prep resin cross-linked with BS three to purify the bait protein VPG PRO and its binding partners. An aliquot of the purified sample was subjected to mass spec analysis for protein identification. The absence of a specific band in the negative control confirms the specificity of the pull down.
The lower band corresponds to the bait protein VPG PRO and the upper band to its binding partner NIB replicate identified by mass spectrometry. Hello, we've just shown you how chemical cross-linking can be used to overcome the limitations associated with Dena and illusion from the strep tact in resin. In addition to purification of protein-protein complexes, the method can be also used to purify protein complexes with nucleic acids including those reversal cross-linked with formaldehyde.
Finally, the method can be used to purify strep tech membrane proteins. Solubilized with detergents. Detergents are needed to keep this hydrophobic proteins in solution.
However, their presence may lead to sample contamination with release strep tane. Even when mild biotin evolution is used, we think that cross-linking of the resin may provide a solution to the sample contamination problem. Thank you for watching and good luck with your experiments.Okay.
