The overall goal of this procedure is to identify post-translational modifications of proteins involved in plant protein complexes first transiently or stably expressed in plana the tags protein of interest. Then extract the total proteins from the plant's tissue, and immuno precipitate the protein of interest. Next, separate the immuno precipitated proteins on an acrylamide gel and extract and digest the proteins from the gel band of interest.
Now submit the protein samples for mass spectrometry. Ultimately, the results of mass spectrometry can detect dynamic changes in post-translational modifications such as phosphorylation. Unlike alternative methods such as the in vitro incubation of kinase with substrate, this method expresses proteins in planter.
So you do not have to know which kinase is responsible for phosphorylation. This method can help answer key questions in the plant biology field, such as how signals are transduced in response to stress, environmental changes, or pathogen recognition. In Addition to the identification of phosphorylation, sites of activated resistance protein complexes, this method can be broadly applied to any post-translational modification of any plant protein complex.
Generally, individuals new to this method will struggle because they're not used to work with large amount of extract and perform more steps of the protocol in a single day. For transient expression, grow the agrobacterium tohin strains to stationary phase. Harvest the agro bacteria by centrifugation at 3000 Gs for five minutes.
Resuspend the pellets and infiltration buffer.Buffer. Now infiltrates. Four week old NCOA hamana plants with agro bacteria manually using a one milliliter needleless syringe or by vacuum with 0.02%surfactant, two to five days post infiltration.
Harvest the infiltrated leaves, leaves, freeze them in liquid nitrogen and store at minus 80 degrees Celsius. Next grinds 20 grams of plant tissue and liquid nitrogen using a mortar and pestle at 80 milliliters of cold buffer C to the 20 grams. Mix well and thaw on ice.
Now homogenize with three bursts of 10 seconds each at full speed. Filter the homogenate and centrifuge at 30, 000 Gs for 30 minutes at four degrees Celsius. Meanwhile to 100 microliters of suitable affinity matrix at 500 microliters of cold blocking buffer D for five minutes at four degrees Celsius and washed three times with one milliliter of cold buffer D.Next, remove the supernat of the leaf extract and pass it through a 0.45 micrometer syringe.
Filter into a new tube on ice. Aliquot the crude extract. For an immuno blot.
Add five XSDS page loading buffer vortex and boil the samples to denature. Add 50 microliters of the affinity matrix, suspended in buffer D to each tube. Incubate with gentle rotation for two hours at four degrees Celsius, centrifuge to pellets the affinity matrix.
Then take an aliquot of the unbound extract for an immuno blot. Discard the rest of the sane, leaving approximately 500 microliters in the bottom of the tube. Resuspend the slurry solution with a wide bore tip.
Pool the mixed slurry solution from both tubes into a single 1.5 milliliter tube. Pulse three times for five seconds using a benchtop centrifuge to precipitate the affinity matrix and remove the supernat after three to five washes with one milliliter of cold buffer D.Remove the excess buffer D with a syringe needle elutes with 200 microliters of the appropriate elution buffer under constant shaking. Pull the three EITs in a single tube.
Then concentrate the proteins by using 30 microliters of absorption resin vortex. Let's stand for five minutes and precipitate the resin, discard the supernat. Add 50 microliters of one XSDS page loading buffer to the precipitated affinity matrix or absorption resin vortex and boil for 10 minutes at 80 to 100 degrees Celsius in a heat block centrifuge.
The boiled affinity matrix are resin for two minutes at 10, 000. GS aliquot five microliters of the supernat for immuno block and run with other fractions onto a 10 centimeter long SDS page. Gel for separation and identification of phosphorylated proteins by mass spectrometry.
Load the remaining 45 microliters onto an SDS page gel stain. The SDS page gel with colloidal kamasi brilliance blue. Then detain the gel with copious washing and water, preferably overnight.
Now with a clean scalpel, excise the band of interest from the gel dice the gel slice into cubes of two to four millimeters and transfer the sample to a tube. Wash the gel pieces with 50%aceto nitrile and 50 millimolar ammonium bicarbonate until de stain. Then pipette off the solution.
Taking care not to remove gel pieces. Dehydrate the sample with 100%aceto nitrile for five minutes, and remove the free liquid. Then to reduce the protein di sulfide bonds at 10 millimolar DTT and incubate for 30 to 45 minutes at 56 degrees Celsius with shaking, remove the free liquid now to alkylate cysteine residues.
Add 55 millimolar chloro Acetamide for 20 to 30 minutes at room temperature in the dark. Remove the free liquid, then wash the gel pieces twice with 50%Aceto nitrile for 10 minutes. Remove free liquid.
Now dehydrate the gel pieces with 100%aceto nitrile for five minutes, and remove free liquid for the Triptych Digest. At 40 microliters of trypsin working solution, allow the gel pieces to rehydrate for 10 minutes at enough digestion buffer to cover gel pieces. Incubate at 37 degrees Celsius overnight to stop the digestion and extract the peptides from the gel pieces at 5%formic acid sonicate for five to 10 minutes.
Then transfer the supernat to a new tube, dry the peptides by lyophilization or a vacuum concentrator, and store the samples at minus 20 degrees Celsius. This experiment purifies the P-R-F-P-T-O kinase complex from nacoa hamana, A transgenic NACOA hamana expressing 35 SPTO was transiently transformed with 35 SPRF flag, and the complex was immuno precipitated with Anti-Flag M two aros. The immuno blots illustrates efficient immunoprecipitation of the targeted PRF protein, both PTO and the interacting effector protein co purified with PRF as indicated by antibody detection and mass spectrometry analysis.
Here, the strategy was to target the PTO protein with Anti-Flag. The strategy was changed. In order to identify PTO phosphorylation sites.
The total amount of PTO protein comprising both the PRF complex and the freeform was immuno precipitated with Anti-Flag M two aros and subjected to SDS page fractionation, ingel triptych digestion, and mass spectrometric analysis resulted in PTO peptides spanning approximately 80%of its sequence. Interestingly, a set of single phosphorylation sites for PTO peptides were identified. Note that double phosphorylation sites were identified only in the presence of either effector protein.
It is important to get enough plant protein quantities in order to identify components of a protein complex and also detect post-translational modification of a protein of interest. Remember that mapping modifications like phosphorylation requires a reasonable amount of protein from mass spectrometry, so at least a faint band should be evident on your gel Following this procedure. Other methods, like I, identification of complex components can be performed in order to answer additional questions like how immune complex activation leads to functional immunity.
Don't forget that working with organic buffers, bacteria and razor BBLs can be extremely hazardous, so take precautions such as wearing gloves and lap coats.
