This work was supported by the National Research Foundation of Korea grant funded by the Korean government (MSIP) (No. 2015R1A2A1A10052663 and No. 2014M1A3A3A02034698).

1. Safety
CAUTION: This protocol uses [&#947;-32P]-ATP to assess the activity of AMPK. Phosphorus-32 is a radioactive isotope, largely emitting beta radiation. Since the size of a beta particle is extremely small, it can easily penetrate clothing and skin. Both external and internal exposure to beta radiation may be harmful to human health, including by causing skin burns and tissue damage.
<ol>
	<li>Carry out all the steps that require the use of [&#947;-32P]-ATP using proper protection, such as acrylic shielding.</li>
	<li>Ensure that all personnel are equipped with electronic personal dosimeters (EPD) to monitor the extent of exposure to harmful radiation during all procedures.</li>
	<li>Handle open-source radiation only after appropriate training and regulatory approval. 
	NOTE: Here, all experiments were conducted after the completion of training on open-source radiation use at the Seoul National University and the approval of the Korean government (nssc.go.kr/nssc/en).</li>
	<li>Dispose of radioactive waste per local regulations. 
	NOTE: Here, directives from the Institute of Environmental Protection &#38; Safety of the Seoul National University were followed. The followings are a list of regulators in the United States and European countries: The United States of America, United States Nuclear Regulatory Commission (http://www.nrc.gov/); United Kingdom, Health and Safety Executive (HSE) (http://www.hse.gov.uk); France, Autorit&#233; de s&#251;ret&#233; nucl&#233;aire (https://www.asn.fr/); and Germany, Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMU) (http://www.bmu.de).</li>
</ol>
2. In Vitro Competitive Kinase Assay
<ol>
	<li>Construction of Competitive Peptides
	<ol>
		<li>Determine the consensus motif phosphorylated by AMPK. 
		NOTE: AMPK recognizes the consensus motif, &#934;-[X,&#946;]-X-X-Ser/Thr-X-X-X-&#934;. In the consensus sequence, &#934; represents hydrophobic amino acids (i.e., valine [V], leucine [L], methionine [M], and isoleucine [I]) and &#946; represents basic amino acids (i.e., lysine [K], arginine [R], and histidine [H])1.</li>
		<li>Select serine or threonine residues from target sequences according to the above consensus motif. Use three putative sites of human Nrf2 (#1, 148-157 comprising Ser153; #2, 330-339 comprising Ser335; and #3, 553-562 comprising Ser558)8.</li>
		<li>Commercially synthesize 10-residue peptides that mimic the putative sites. Obtain the synthesized product as a lyophilized powder with a purity greater than 98% for each peptide.</li>
		<li>Dissolve the peptides using a kinase buffer to achieve a concentration of 71.667 g/L. If a peptide is not soluble in a kinase buffer, dissolve it in 20% DMSO. If it remains insoluble, a peptide mimicking the putative site may be extended to increase solubility.</li>
	</ol>
	</li>
</ol>
<ol start="2">
	<li>In Vitro Competitive AMPK Kinase Assay
	<ol>
		<li>Prepare 5&#215; kinase buffer as follows: 100 mM HEPES, pH 7.4; 25 mM MgCl2; 5 mM EGTA; 5 mM DTT; 125 mM &#946;-glycerophosphate (serine/threonine phosphatase inhibitor); 5 mM Na3VO4 (tyrosine phosphatase inhibitor); 0.5% (v/v) Protease Inhibitor Cocktail Set III, 1 mM cold ATP; and 500 &#181;M AMP. 
		NOTE: AMP is an essential component of the kinase buffer to test AMPK activity. The buffer can be used to measure the activity of both serine/threonine kinases and tyrosine kinases.</li>
		<li>Thaw the following recombinant protein and synthetic peptides on ice: AMPK; Nrf2; and oligopeptides #1, #2, and #3.</li>
		<li>Prepare a reaction buffer on ice: 0.15 &#181;g of AMPK, 0.4 &#181;g of Nrf2 (~4 pmol), 0.43 mg of oligopeptide (~300 nmol), and 6 &#181;L of 5&#215; kinase buffer. Adjust the final volume to 30 &#181;L with sterile distilled water (DW). 
		NOTE: The addition of [&#947;-32P]-ATP after preparing the reaction buffer may reduce the radioactive signal due to kinase autophosphorylation.</li>
	</ol>
	</li>
	<li>Running the Reactants 
	NOTE: All the processes below must be carried out under protection with an acrylic shield, rack, or block, as well as appropriate personal protective equipment.
	<ol>
		<li>Set heating blocks to 30 &#176;C before starting the experiment.</li>
		<li>Add 1 &#181;L of [&#947;-32P]-ATP (1 &#181;Ci) to the reaction tubes on ice. Mix the reaction buffer by pipetting up and down several times. 
		NOTE: The radioactivity of 32P has a short half-life of approximately 14 days9. Adjust the volume by considering the half-life (e.g., after one month from the production of [&#947;-32P]-ATP, add 4 &#181;L of [&#947;-32P]-ATP to make 1 &#181;Ci).</li>
		<li>Incubate the sample in the heating block at 30 &#176;C for 15-30 min. During this step, prepare 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Adjust the percentage of the gel according to the size of the target protein.</li>
		<li>Stop the kinase reaction by adding 3 &#181;L of 10&#215; SDS sample buffer (10% (v/v) glycerol, 20% (w/v) SDS, 15.42% (w/v) dithiothreitol, 90% (v/v) 0.5 M Tris-HCl (pH 6.8), and 0.02% (w/v) bromophenol blue). Mix the reaction buffer by pipetting up and down several times.</li>
		<li>Run the samples in a 7.5% SDS-PAGE gel at 70 V for 20 min and continuously at 140 V for 1 h. 
		NOTE: The bromophenol blue line at the end of the gel contains extremely high radioactive signals. It is recommended to remove the gel below the blue line before proceeding to the next step to reduce background signals.</li>
		<li>After SDS-PAGE, gently remove the gel from the caster. Place the gel in a glass tube for the next step.</li>
		<li>Fix the gel with fixation buffer for 20 min (50% methanol and 10% glacial acetic acid).</li>
		<li>Gently move the gel onto filter paper and cover it with a transparent wrapper. Note that the gel may be easily torn at this step. Dry the gel with a vacuum gel dryer at 80 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Visualization
	<ol>
		<li>Expose the gel to either a phosphor screen or an X-ray film overnight (usually for approximately 16 h). 
		NOTE: A phosphor screen can be reused after exposure to the extra-bright light. When an X-ray film is used, expose the gel over two days. X-ray films have lower sensitivities than phosphor screens.</li>
		<li>Scan the phosphor screen with a phosphor imager. Export the image as a high-resolution TIFF or bitmap file.</li>
		<li>After visualization, move the gel to a glass or plastic container for Coomassie brilliant blue (CBB) staining.</li>
		<li>Wash the gel with DW. Discard the DW and stain the gel with the staining reagent for 1 h.</li>
		<li>Discard the reagent and destain the gel with DW for 1 h or overnight. Place the gel between transparent plastic films (or the equivalent) to scan with an office scanner.</li>
	</ol>
	</li>
</ol>
3. In Vitro Kinase Assay Using a Site-specific Mutant
<ol>
	<li>Site-Directed Mutagenesis of pGEX-Nrf2
	<ol>
		<li>Mutagenic Primer Design 
		NOTE: Designing proper mutagenic primers is critical for the successful outcome of mutagenesis. The following are some considerations for designing mutagenic primers. Create a primer consisting of 25 to 45 bases, with the desired mutation in the middle of the primer. The primer should have 40-60% GC content and terminate with one or more G or C bases. Ensure that the Tm of the primer is equal to 78 &#176;C or greater according to the formula [Tm= 81.5 + 0.41(% GC) - (675/length of bases) - % mismatch]. 
		NOTE: Purify the primer because its purity is important for mutation efficiency. There are several websites for designing mutagenic primers.
		<ol>
			<li>Open the primer design program for &#34;designing primers for site-directed mutagenesis&#34; (http://www.genomics.agilent.com/primerDesignProgram.jsp).</li>
			<li>Paste human Nrf2 DNA sequence (Access No. NM_006164.4) and select the &#34;upload translated&#34; button.</li>
			<li>Select the serine 558 residue from the translated amino acid sequence to be changed to alanine. Using the mutagenic primer, substitute the codon for serine from agc to gcc, for alanine.</li>
			<li>Consider primer length, Tm, and the GC content. 
			NOTE: The mutagenic primer is finally designed as 5&#39;-ctactgaaaaaacaactc gccaccttatatctcgaag-3&#39;.</li>
		</ol>
		</li>
		<li>Reaction for Mutant Strand Synthesis using Polymerase Chain Reaction (PCR)
		<ol>
			<li>Prepare the PCR reaction mixture on ice: 10&#215; reaction buffer, 50 ng of pGEX-GST-Nrf2, 125 ng of forward primer, 125 ng of reverse primer, 1 &#181;L of dNTP mix (10 mM each), 2.5 U Pfu DNA polymerase, and sterile DW to 50 &#181;L.</li>
			<li>Run the PCR for pGEX-GST-Nrf2-mutant strand synthesis with the following cycling program: 1 cycle at 95 &#176;C for 30 s; 18 cycles at 95 &#176;C for 30 s, at 55 &#176;C for 1 min, and at 68 &#176;C for 90 s; and 1 cycle at 68 &#176;C for 5 min. 
			NOTE: Depending on the plasmid length, the cycling parameters may be changed.</li>
			<li>Place it on ice for 2 min to cool the reactants for the next step. Alternatively, store them at 4 &#176;C overnight.</li>
		</ol>
		</li>
		<li>Dpn I Digestion and the Selection of a Mutant Plasmid
		<ol>
			<li>Add 1 &#181;L of Dpn I restriction enzyme (10 U/&#181;L) to each of the reactants after cooling. Mix thoroughly and gently by pipetting and centrifuge for 1 min.</li>
			<li>Incubate at 37 &#176;C for 2 h to digest parental pGEX-GST-Nrf2 double-stranded DNA.</li>
			<li>Gently thaw DH5&#160;&#945; supercompetent cells on ice.</li>
			<li>Add 50 &#181;L of the reactant to 150 &#181;L of pre-chilled DH5 &#945; supercompetent cells and incubate on ice for at least 30 min.</li>
			<li>Transfer the tubes to a heating block preheated to 42 &#176;C and leave them for 90 s. Place them on ice for 2 min.</li>
			<li>Add pre-warmed lysogeny broth (LB) to the transformed DH5&#160;&#945; cells and incubate at 37 &#176;C for 1 h with shaking at 180 rpm.</li>
			<li>Spread the transformed cells on LB-ampicillin agar plates and incubate overnight at 37 &#176;C. 
			NOTE: Ampicillin is used for selection since the plasmid GEX-GST-Nrf2 has the ampicillin resistance marker. Use appropriate antibiotics, depending on the resistance marker of the mutagenized plasmid.</li>
			<li>Pick a single colony from the plate, transfer it to 5 mL of LB-ampicillin, and incubate overnight it at 37 &#176;C with shaking at 180 rpm. For verification of the mutagenic sequence, evaluate at least three colonies.</li>
			<li>Extract DNA using a DNA miniprep kit for DNA sequence analysis; follow the manufacturer&#39;s protocol.</li>
			<li>Verify the mutagenic DNA sequences using an automatic DNA sequence analyzer according to the manufacturer&#39;s protocol. Use at least 200 ng of the DNA construct for the verification of mutant sequences.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Purification of Recombinant GST-Nrf2 Protein
	<ol>
		<li>Expression of Mutagenic GST-Nrf2 Fusion Protein in Escherichia coli
		<ol>
			<li>Transform the mutagenic pGEX-GST-Nrf2 plasmid into BL21 cells. Inoculate a single colony in 25 mL of LB broth containing ampicillin (100 &#181;g/mL) and incubate at 37 &#176;C on a rotator overnight.</li>
			<li>Inoculate the cultured cells in 100 mL of LB broth containing ampicillin (100 &#181;g/mL) at a 1:100 dilution ratio.</li>
			<li>Incubate at 37 &#176;C on a rotator until the absorbance at 600 nm reaches around 0.4-0.8 (approximately 4 h).</li>
			<li>Add 100 &#181;L of 1 M IPTG to 100 mL of cultured cells to prepare a final concentration of 1 mM IPTG.</li>
			<li>Incubate the cells at 30 &#176;C on a rotator for 6 h or overnight for protein expression.</li>
			<li>Centrifuge the cells at 5,000 x g for 20 min and resuspend the cell pellets with 1 mL of bacterial lysis buffer (25 mM HEPES, 5 mM EDTA, 2 mM DTT, 0.1% CHAPS, 1 &#181;g/mL pepstatin, 0.5 &#181;g/mL leupeptin, and 1 mM PMSF) on ice.</li>
			<li>Lyse the cell pellets using an ultrasonic homogenizer with 2-s intervals for 2 min at 50% of the maximum power output.</li>
			<li>Centrifuge the cells at 12,000&#160;&#215; g for 15 min at 4 &#176;C and collect the supernatant.</li>
		</ol>
		</li>
		<li>Purification of Recombinant GST-Nrf2 Protein
		<ol>
			<li>Prepare 200 &#181;L of 50% (v/v) glutathione-agarose beads and wash with 1 mL PBS (pH 7.3; 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4). Collect the beads by centrifugation at 500&#160;&#215; g for 1 min. Repeat the washing step three times.</li>
			<li>Add the cell lysate to the prepared 200 &#181;L of 50% (v/v) glutathione-agarose beads and incubate at 4 &#176;C on an end-over-end rotator for 2 h.</li>
			<li>Collect the glutathione-agarose beads combined with GST-Nrf2 protein by centrifugation at 500 x g for 1 min. Remove the supernatant using a 1 mL syringe with a 31 G needle.</li>
			<li>Wash the beads with 1 mL of PBS (pH 7.3), as in step 1.</li>
			<li>Add 500 &#181;L of elution buffer (50 mM Tris-HCl and 10 mM reduced glutathione, pH 8.0) to the glutathione-agarose beads combined with GST-Nrf2. Incubate at 4 &#176;C on an end-over-end rotator for 30 min.</li>
			<li>Centrifuge at 500 x g for 1 min and collect the supernatant.</li>
			<li>Again add 300 &#181;L of elution buffer to the beads and repeat steps 3.2.2.5-3.2.2.6 twice.</li>
		</ol>
		</li>
		<li>Confirmation of the Purified GST-Nrf2 Protein
		<ol>
			<li>Prepare 5 &#181;L of the sample for visualization of protein quantity with 0.5 &#181;g of standard protein BSA.</li>
			<li>Run the samples in a 7.5% SDS-PAGE gel at 70 V for 20 min and then at 140 V for 60 min.</li>
			<li>Stain the gel with 0.1% CBB staining solution (0.1 g of CBB R250, 40 mL of 99% methanol, 10 mL of glacial acetic acid, and 50 mL of distillated H2O) for 2 h and destain (30 mL of methanol, 10 mL of glacial acetic acid, and 60 mL of distilled H2O) until the bands are clearly shown.</li>
			<li>Place the destained gel on filter paper and cover with a transparent wrapper. Dry the gel with a vacuum gel dryer at 80 &#176;C for 1 h.</li>
			<li>Determine the quantity of mutant GST-Nrf2 protein with densitometry for the in vitro AMPK kinase assay.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In Vitro AMPK Activity Assay with a Site-Specific Mutant 
	NOTE: The in vitro AMPK kinase assay is performed according to steps 2.2-2.4, using 0.4 &#181;g of purified wildtype GST-Nrf2 or the GST-Nrf2-S558A mutant without peptide inhibitors. Follow all the radiation safety guidelines described in step 1.
	<ol>
		<li>Prepare a reaction buffer on ice with 0.15 &#181;g of AMPK, 0.4 &#181;g of mutant or wildtype GST-Nrf2, and 6 &#181;L of 5 kinase buffer. Fill up to 30 &#181;L with sterile DW.</li>
		<li>Add 1 &#181;L of [&#947;-32P]-ATP (1 &#181;Ci/&#181;L) to the reaction tubes on ice. Mix the reaction buffer by pipetting up and down several times and centrifuge.</li>
		<li>Incubate the samples in a heating block at 30 &#176;C for 30 min.</li>
		<li>Stop the kinase reaction by adding 3 &#181;L of 10&#215; SDS sample buffer.</li>
		<li>Run in a 7.5% SDS-PAGE gel at 70 V for 20 min and then at 140 V for 1 h.</li>
		<li>Fix the gel with the fixation buffer for 20 min, move it onto the filter paper, and cover the gel with a transparent wrapper.</li>
		<li>Dry the gel with the vacuum gel dryer at 80 &#176;C for 1 h and then expose it overnight to either a phosphor screen or an X-ray film.</li>
		<li>Scan the phosphor screen with a phosphor imager and move the gel to a glass tube for CBB staining after visualization according to step 2.4.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hardie, D. G., Schaffer, B. E., Brunet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK:+An+energy-sensing+pathway+with+multiple+inputs+and+outputs.">AMPK: An energy-sensing pathway with multiple inputs and outputs.</a> Trends Cell Biol. 26, 190-201 (2016).</li><li>Gwinn, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK+phosphorylation+of+raptor+mediates+a+metabolic+checkpoint.">AMPK phosphorylation of raptor mediates a metabolic checkpoint.</a> Mol Cell. 30, 214-226 (2008).</li><li>Huang, H. C., Nguyen, T., Pickett, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Nrf2+at+Ser-40+by+protein+kinase+C+regulates+antioxidant+response+element-mediated+transcription.">Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.</a> J Biol Chem. 277, 42769-42774 (2002).</li><li>Niture, S. K., Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant-induced+modification+of+INrf2+cysteine+151+and+PKC-delta-mediated+phosphorylation+of+Nrf2+serine+40+are+both+required+for+stabilization+and+nuclear+translocation+of+Nrf2+and+increased+drug+resistance.">Antioxidant-induced modification of INrf2 cysteine 151 and PKC-delta-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance.</a> J Cell Sci. 122, 4452-4464 (2009).</li><li>Jain, A. K., Jaiswal, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GSK-3beta+acts+upstream+of+Fyn+kinase+in+regulation+of+nuclear+export+and+degradation+of+NF-E2+related+factor+2.">GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2.</a> J Biol Chem. 282, 16502-16510 (2007).</li><li>Steen, H., Jebanathirajah, J. A., Rush, J., Morrice, N., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+analysis+by+mass+spectrometry:+myths,+facts,+and+the+consequences+for+qualitative+and+quantitative+measurements.">Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.</a> Mol Cell Proteomics. 5, 172-181 (2006).</li><li>Dephoure, N., Gould, K. L., Gygi, S. P., Kellogg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+analysis+of+phosphorylation+sites:+a+quick+guide+for+cell+biologists.">Mapping and analysis of phosphorylation sites: a quick guide for cell biologists.</a> Mol Biol Cell. 24, 535-542 (2013).</li><li>Joo, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPK+facilitates+nuclear+accumulation+of+Nrf2+by+phosphorylating+at+Serine+550.">AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at Serine 550.</a> Mol Cell Biol. 36, 1931-1942 (2016).</li><li>Sinclair, W. K., Holloway, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-lives+of+some+radioactive+isotopes.">Half-lives of some radioactive isotopes.</a> Nature. 167 (4244), 365 (1951).</li><li>Chang, B. Y., Chiang, M., Cartwright, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+Src+and+RACK1+is+enhanced+by+activation+of+protein+kinase+C+and+tyrosine+phosphorylation+of+RACK1.">The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1.</a> J Biol Chem. 276, 20346-20356 (2001).</li><li>Smith, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RACK1+interacts+with+filamin-A+to+regulate+plasma+membrane+levels+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.</a> Am J Physiol Cell Physiol. 305 (1), C111-C120 (2013).</li></ol>

The authors have nothing to disclose.

As a simple and convenient way to assess the authenticity of the predicted phosphorylation sites mediated by AMPK, here we describe an in vitro kinase assay that can be used to discover a specific phosphorylation site using competitive peptides and to verify it using a site-specific mutant. The representative data obtained from the in vitro competitive AMPK activity assay matched the results from an assay using a site-directed mutant protein, indicating that the peptide competition assay is a useful tool for determining a phosphorylation site. This method was also used in the study identifying the specific site phosphorylated by PKC&#948;, which phosphorylates Nrf2 at serine 403. Since peptides competitively bind to a protein, the principle may also be applied to an in vitro GST pull-down assay to identify a binding motif10,11. Moreover, this peptidomimetic method may also be applicable to the identification of other post-translational modifications, such as acetylation at lysine residues.
Using this protocol, it may be difficult to dissolve some hydrophobic peptides. If a peptide is insoluble in a kinase buffer, dissolving it in 20% DMSO may be helpful. To dissolve extremely hydrophobic peptides, first attempt to dissolve it in 100% DMSO and then dilute the solution with a kinase buffer. Sonication may also be of help in dissolving the peptides. If it remains insoluble, a peptide mimicking the putative site should be extended to increase the solubility.
The protocol introduced in this paper describes an &#34;in vitro system&#34; in which a purified kinase and substrate are reacted. In the in vitro system, small differences in the concentration of competitive peptides will not affect the results unless the concentration is lower than the substrate protein. The result obtained from an in vitro experiment includes the inherent possibility of being an artefactual event produced by artificial proximity and the high concentration of reactants. To eliminate this prospect, the interpretation of the data should be accompanied by the outcomes from cell-based experiments. Another possibility for obtaining false-positive results using peptides may be due to alterations in substrate conformation and/or kinase recognition. To exclude this possibility, it is necessary to do confirmation experiments with mutant proteins.
On the other hand, false-negative data can also be produced from this system. Many kinases act as protein complexes within the cellular context and require co-factors for full functionality. AMPK is a representative protein kinase complex composed of AMPK&#945;, AMPK&#946; and AMPK&#947; subunits, and the binding of AMP to the AMPK&#947; subunit is necessary for the activation of the kinase. Indeed, AMPK did not phosphorylate Nrf2 in an AMP-free kinase buffer (data not shown). Thus, it should be noted that AMP must be dissolved in the kinase reaction buffer for a successful in vitro assay.
The use of mass spectrometry for the identification of phosphorylation sites, the most popular method, has several technical limitations, often generating false-negative signals by the facile loss of phosphoric acid during the fragmentation process7. A peptide microarray method can be the second option to search for phosphorylation sites. However, this method is considered time-consuming and expensive. As an alternative method for screening putative phosphorylation sites, the competitive peptide method may be a convenient and inexpensive way to gain insight into the nature of a putative phosphorylation site.

Protein phosphorylation at a specific residue plays a significant role in a wide range of cellular processes. Thus, an understanding of signaling networks requires the identification of specific phosphorylation sites. In addition, the phosphorylation site determines the effect on protein function because individual domains within a protein possess different structures and functions. The activity of nuclear factor erythroid 2-related factor 2 (Nrf2), a key antioxidant transcription factor, is bi-directionally regulated through phosphorylation at different sites. Our studies have focused on the kinases that catalyze the phosphorylation of Nrf2. The stress response of Nrf2 against oxidative challenge occurs rapidly, mainly through phosphorylation at serine 40 and mediated by protein kinase C (PKC)-&#948;, which activates Nrf23,4. Conversely, Fyn catalyzes the inhibitory phosphorylation of Nrf2 at tyrosine 568 for tight control of the activity5.
The most common method used to discover phosphorylation sites is liquid chromatography/mass spectrometry (LC/MS) analysis. Rapid and highly sensitive phosphorylation site mapping data can be generated this way; however, it has several technical limitations, often generating false-negative signals. Poor sequence coverage frequently occurs in LC/MS analysis. To identify phosphorylation sites, information on the maximal amino acid coverage of a protein is required6. Proteolysis of the protein of interest with several proteases during the digestion step may be of help in improving sequence coverage. Another obstacle for the identification of phosphorylation residues is the facile loss of phosphoric acid that is frequently observed for serine- and threonine-phosphorylated peptides6,7. Labile phosphate moieties are often released from phosphopeptides during the fragmentation process7. The second option when searching for phosphorylation sites is using a peptide microarray method. It is possible to screen for the kinase target sites using a microarray chip containing peptide fragments derived from a protein of interest. However, due to the equipment requirement for the production and detection of a microarray chip, the peptide microarray method is considered time-consuming and expensive.
To overcome these challenges, an in vitro oligopeptide-competing kinase assay can be used for a protein kinase with known recognition motifs. If the consensus recognition motif of a kinase is established, putative phosphorylation sites of a candidate substrate can be predicted, and the authenticity of the sites can be validated. The most convincing method for this procedure is to show the abrogation of phosphorylation in a mutant protein in which the predicted residue is substituted with a non-phosphorylatable amino acid (i.e., serine or threonine to alanine; tyrosine to phenylalanine). However, the production and isolation of mutant proteins is time-consuming. As an alternative at the initial stage of research, the competitive peptide kinase assay is straightforward and convenient. Here, we describe a protocol for an in vitro peptide competition assay and for the validation of the phosphorylation site.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>208337</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Calbiochem, Nottingham, UK</td>
			<td>539134</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1752</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPK</td>
			<td>Upstate Biotechnology, Lake Placid, NY</td>
			<td>14-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Nrf2 (WT)</td>
			<td>Abnova, Taipei City, Taiwan</td>
			<td>H00004780-P01</td>
			<td></td>
		</tr>
		<tr>
			<td>[&#947;-32P]-ATP</td>
			<td>PerkinElmer Life and Analytical Sciences, Waltham, MA</td>
			<td>NEG502A</td>
			<td></td>
		</tr>
		<tr>
			<td>EZblue staining reagent</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G1041</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu turbo DNA polymerase</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td>600250</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Agilent Technologies, Santa Clara, CA</td>
			<td>200415-51</td>
			<td>Avoid multiple thaw and freezing cycle</td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Duchefa Biochemie BV, Haarlem, Netherlands</td>
			<td>L1704</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix, Santa Clara, CA</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose LE</td>
			<td>iNtRON Biotechnology, Sungnam, South Korea</td>
			<td>32034</td>
			<td></td>
		</tr>
		<tr>
			<td>HiYield Plus Gel/PCR DNA Mini Kit</td>
			<td>Real Biotech Corporation, Taipei, Taiwan</td>
			<td>QDF100</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>161-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Bovogen Biologicals, Victoria, Australia</td>
			<td>BSA100</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose 4B</td>
			<td>GE Healthcare, Marlborough, MA</td>
			<td>17-0756-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid glacial</td>
			<td>Duksan pure chemicals, Ansan, South Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl alcohol</td>
			<td>Daejung Chemicals &#38; Metals, Siheung, South Korea</td>
			<td>5558-4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare, Marlborough, MA</td>
			<td>28-9558-09</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE kit</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>165-178</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>165-1746</td>
			<td></td>
		</tr>
		<tr>
			<td>Dancing shaker</td>
			<td>FINEPCR, Seoul, Korea</td>
			<td>CR300</td>
			<td>The machine is needed for washing step</td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator/shakers</td>
			<td>N-BIOTEK, GyeongGi-Do, Korea</td>
			<td>NB-205L</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuges</td>
			<td>LABOGENE, Seoul, Korea</td>
			<td>1730R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography columns</td>
			<td>Bio-Rad Laboratories, Hercules, CA</td>
			<td>732-1010</td>
			<td></td>
		</tr>
	</tbody>
</table>

oligopeptide competition assay for phosphorylation site determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Figure 1 and Figure 2 demonstrate the outcomes from repeated experiments in the previously reported paper8. Three different 10-residue oligopeptides mimicking the putative AMPK target sites (#1, 148-157 comprising Ser153; #2, 330-339 comprising Ser335; and #3, 553-562 comprising Ser558) were synthesized and used as competitors in the in vitro kinase assay. The phosphorylation of Nrf2 by AMPK was greatly diminished in the presence of oligopeptide #3 (Figure 1). Next, a site-directed mutagenesis assay was performed to validate the result of the peptidomimetic experiment. Given the inhibitory effect of oligopeptide #3 on the AMPK-mediated phosphorylation of Nrf2 (Figure 1), we generated a S558A-Nrf2 mutant. The phosphorylation levels were compared between the wildtype Nrf2 and the S558A mutant using the in vitro AMPK kinase assay. The single amino acid substitution of Nrf2 (Ser&#8594;Ala at 558) prevented AMPK from phosphorylating Nrf2, indicating that AMPK directly phosphorylates Nrf2 at Ser558 (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55708/55708fig1.jpg" /> 
Figure 1: In vitro competitive AMPK kinase assay. In vitro kinase assays were done in the presence of the indicated oligopeptides (#1, 148-157 comprising Ser153; #2, 330-339 comprising Ser335; and #3, 553-562 comprising Ser558).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55708/55708fig2.jpg" /> 
Figure 2: In vitro AMPK kinase assay using a site-specific mutant protein. In vitro kinase assays were done in the presence of wildtype GST-Nrf2 or its S558A mutant.

Watch this Scientific Journal Video about Oligopeptide Competition Assay for Phosphorylation Site Determination at JoVE.com

Oligopeptide Competition Assay for Phosphorylation Site Determination

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

oligopeptide-competition-assay-for-phosphorylation-site-determination

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.4K Views.  Seoul National University. Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Video: Oligopeptide Competition Assay for Phosphorylation Site Determination

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning because it is involved in microtubule assembly and stabilization. Besides neurons, tau is expressed in human breast, prostate, gastric, colorectal, and pancreatic cancers where it shows nearly similar structure and exerts similar functions as the neuronal tau. The amount of tau and its phosphorylation can change its function as a stabilizer of microtubules, and lead to the development of paired helical filaments in different neurodegenerative disorders, such as Alzheimer&#39;s disease. Determining the phosphorylation state of tau and its microtubule-binding characteristics is important. In addition, examining the intracellular localization of tau is important in different diseases. This manuscript details standard protocols for measuring tau phosphorylation and tau binding to microtubules in colorectal cancer cells with or without curcumin and LiCl treatment. These treatments can be used to stop cancer cell proliferation and development. Intracellular localization of tau is examined by using immunohistochemistry and confocal microscopy while using low amounts of antibodies. These assays can be used repetitively for screening compounds that affect tau hyperphosphorylation or microtubule binding. Novel therapeutics used for different tauopathies or related anticancer agents can potentially be characterized using these protocols.

This manuscript describes standard protocols for measuring tau hyperphosphorylation, measuring tau binding to microtubules, and localization of intracellular tau following drug treatments. These protocols can be used repetitively for screening drugs or other compounds that target tau hyperphosphorylation or microtubule binding.

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Summary

Explore More Videos

Chapters in this video

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Functional Protein Regions Through Chimeric Protein Construction

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli