Name: in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions
Uploaded: 2019-07-17T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions at JoVE.com

We thank P. Jeremy Wang (University of Pennsylvania) for helpful edits and discussions. We also thank Sigrid Eckardt for language editing. K. Z. was supported by National Key R&#38;D Program of China (2016YFA0500902, 2018YFC1003500) and National Natural Science Foundation of China (31771653). L. Y. was supported by National Natural Science Foundation of China (81471502, 31871503) and Innovative and Entrepreneurial Program of Jiangsu Province.&#160;J. N. was supported by Zhejiang Medical Science and Technology Project (2019KY499).&#160;M. L. was supported by grants of National Natural Science Foundation of China (31771588) and the 1000 Youth Talent Plan.

1. Protein preparation
<ol>
	<li>MEIOB and MOV10 expression constructs
	<ol>
		<li>Generate cDNA expression constructs encoding mouse MEIOB-A, C, and E (Figure 1A) and MOV10.
		<ol>
			<li>Set up the polymerase chain reaction (PCR) reactions for each fragment. Mix 1 &#956;L of mouse cDNA (from C57BL/6 mouse testis), 1 &#956;L of dNTP, 2 &#956;L of 10 &#956;M forward primer, 2 &#956;L of 10 &#956;M reverse primer, 1 &#956;L of DNA polymerase, 25 &#956;L of 2x PCR buffer, and 18 &#956;L of double distilled H2O (ddH2O) in a final volume of 50 &#956;L. 
			NOTE: The primers for the amplification of Meiob and Mov10 gene fragments are listed in Table 1.</li>
			<li>Perform PCR reactions using the following programs: 95 &#176;C for 5 min, 35 cycles of heating at 95 &#176;C for 15 s, annealing at 64 &#176;C for 15 s, extension at 72 &#176;C for 20 s (2 min for extending full length MOV10), and final extension at 68 &#176;C for 7 min. 
			NOTE: Use primer pair MEIOB-E (F1) and MEIOB-E-mut (R1) and MEIOB-E-mut (F2) and MEIOB-E (R2) to amplify two segments that contain the mutant sequence within an overlapping sequence at the 3&#39;&#160;and 5&#39;&#160;ends, respectively, to generate a mutant PCR template.</li>
		</ol>
		</li>
		<li>Analyze the amplified PCR DNA by gel electrophoresis, cut the band of required size from the gel quickly under a UV lamp, and place into a centrifuge tube. 
		NOTE: The expected product sizes visible on the agarose gel for MEIOB-A is 536 bp, MEIOB-C is 296 bp, MEIOB-E are 312 bp and 229 bp, and MOV10 is 3015 bp.</li>
		<li>Purify the PCR DNA with a gel extraction kit following the manufacturer&#39;s protocol.
		<ol>
			<li>Add an equal volume of dissolving buffer into the centrifuge tube from step 1.1.2 and melt gel in a 50-55 &#176;C water bath for 5-10 min, ensuring that the gel pieces melt completely. Centrifuge briefly to collect any droplets from the wall of the tube. 
			NOTE: The mass/volume concentration of the gel and the dissolving buffer is 1 mg/&#956;L.</li>
			<li>Place the adsorption column in the collection tube, transfer the solution containing the dissolved gel fragment to the adsorption column, and centrifuge at 12,000 x g for 2 min.</li>
			<li>Discard the filtrate at the bottom of the collection tubes. Add 600 &#956;L of the wash buffer to the column, centrifuge at 12,000 x g for 1 min, and discard the filtrate.</li>
			<li>Repeat step 1.1.3.3 once.</li>
			<li>Place the column back into the collection tube, and centrifuge at 12,000 x g for 2 min to remove all the remaining wash buffer.</li>
			<li>Place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 50 &#956;L of ddH2O to the center of the adsorption column and centrifuge at 12,000 x g for 1 min. Measure the DNA concentration of the eluate using spectrophotometer.</li>
		</ol>
		</li>
		<li>Restriction digestion of plasmids
		<ol>
			<li>Digest pGEX-4T-1 vector with BamHI and NotI. To do so, mix 4 &#956;g of pGEX-4T-1 vector, 5 &#956;L of 10x digest buffer, 1 &#956;L of BamHI, and 1 &#956;L of NotI and ddH2O to a final reaction volume of 50 &#956;L. Incubate at 37 &#176;C for 2 h.</li>
			<li>Digest pRK5 vector with BamHI and XhoI by mixing 4 &#956;g of pRK5 vector, 5 &#956;L of 10x digest buffer, 1 &#956;L of BamHI, and 1 &#956;L of XhoI and ddH2O to a final reaction volume of 50 &#956;L. Incubate at 37 &#176;C for 2 h.</li>
		</ol>
		</li>
		<li>Analyze the vector DNA by gel electrophoresis, cut the desired size band from the gel quickly with a scalpel under a UV lamp and place it into a centrifuge tube.</li>
		<li>Purify the vector DNA with a gel extraction kit as 1.1.3 following the manufacturer&#39;s instruction. 
		NOTE: The length of linearized plasmids: pGEX-4T-1, 4.4 kb; pRK5, 4.7 kb.</li>
		<li>Set up a standard recombinant ligation reaction by combining 0.03 pmol of linearized vector, 0.06 pmol of cDNA fragment, 2 &#956;L of ligase, and 4 &#956;L of 5x ligase buffer and ddH2O in a final reaction volume of 10 &#956;L. 
		NOTE: Clone MEIOB-A, C, and E into a pGEX-4T-1 vector and MOV10 into a pRK5 vector.</li>
		<li>Incubate the mixture at 37 &#176;C for 30 min, and then cool the reaction immediately for 5 min on ice. Transform MEIOB recombinant plasmids into BL21 bacteria and MOV10 recombinant plasmids into DH5&#945;&#160;bacteria. 
		NOTE: Verify all recombinant constructs by Sanger sequencing.</li>
		<li>Prepare glycerol stocks of bacterial cultures containing verified recombinant plasmids by adding an equal volume of 50% glycerol to liquid cultures, and store at -80 &#176;C. 
		NOTE: For each subsequent experiment, streak out bacteria from glycerol stocks onto a fresh agar plate and use a single colony for the expansion as described in step 1.2.</li>
	</ol>
	</li>
	<li>MEIOB protein extracts from bacteria
	<ol>
		<li>Pick one colony of each BL21 strain transfected with the empty or recombinant pGEX-4T-1 plasmid verified by sequencing and inoculate in 3 mL LB containing 100 &#181;g/mL ampicillin. Grow overnight at 37 &#176;C with shaking at 220 rpm.</li>
		<li>Inoculate 300 mL LB containing 100 &#181;g/mL ampicillin from 3 mL overnight culture (from step 1.2.1). Grow the cultures with shaking at 37 &#176;C for 2 h till OD600 reaches 0.5-1.0.</li>
		<li>Induce the protein expression by adding isopropyl beta-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM. Incubate the cultures for an additional 3 h with shaking at 180 rpm and 18 &#176;C.</li>
		<li>Centrifuge the bacterial culture at 3,500 x g and 4 &#176;C for 20 min.</li>
		<li>Resuspend the pellet in 20 mL of ice-cold Dulbecco&#39;s phosphate buffered saline (DPBS) buffer. Sonicate the bacterial suspension on ice for 25 cycles in short 10 s bursts (output power 20%) followed by 2-3 s resting on ice.</li>
		<li>Centrifuge the lysate at 12,000 x g and 4 &#176;C for 15 min. Transfer all the supernatant to a fresh tube.</li>
		<li>Pre-wash beads.
		<ol>
			<li>Add 300 &#956;L of glutathione-sepharose beads to a fresh 15&#160;mL tube and wash the beads with 10 mL of ice-cold PBS buffer.</li>
			<li>Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads and discard the wash solution.</li>
		</ol>
		</li>
		<li>Add the lysate to the washed beads and incubate at 4 &#176;C for 2 h. Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads. Rinse the beads in 10 mL of ice-cold PBS 8x.</li>
		<li>Elute the beads with 1 mL of the elution buffer (10 mM glutathione in 50 mM Tris-HCl at pH 8.0) 6x, incubating at 4 &#176;C for 10 min prior to each elution step.&#160;Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads.&#160;Collect and pool the 6 fractions.</li>
		<li>Transfer the eluted proteins into a centrifugal filter and concentrate by centrifugation at 7,500 x g to obtain a final volume of 100-200 &#956;L.</li>
	</ol>
	</li>
	<li>MOV10 protein extracts from HEK293T cells
	<ol>
		<li>Transiently express the MOV10 proteins in cultured HEK293T cells.
		<ol>
			<li>Prepare MOV10-pRK5 plasmid at a concentration &#62;500 ng/&#956;L.</li>
			<li>Seed HEK293T cells in 15 cm dishes. When the cell density reaches ~70%-90%, replace the cell culture medium with fresh Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.</li>
			<li>For one transfection, dilute 60 &#956;g of MOV10-pRK5 plasmid DNA in 3 mL of the reduced serum medium, then add 120 &#956;L of the transfection enhancer reagent, and mix well.</li>
			<li>In a separate tube dilute 90 &#956;L of the transfection reagent with 3 mL of reduced serum medium (without penicillin-streptomycin) and mix well.</li>
			<li>Add the diluted DNA to each tube of diluted transfection reagent. Incubate at room temperature for 15 min.</li>
			<li>Add the transfection mixture to the cell culture, and culture cells for ~36-48 h.</li>
		</ol>
		</li>
		<li>After 36-48 h, collect cells from each plate in a 50 mL tube. Centrifuge at 500 x g for 5 min at 4 &#176;C. Wash each pellet with 10 mL of ice-cold PBS, and collect cells by centrifugation at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the pellet in 3 mL of cell lysis buffer containing complete ehylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail. Incubate for 30 min on ice. Centrifuge the lysate at 20,000 x g and 4 &#176;C for 20 min.</li>
		<li>Add 100 &#956;L of anti-FLAG magnetic beads per dish of cells in a 1.5 mL tube.</li>
		<li>Wash the magnetic beads 2x with K150 buffer (50 mM HEPES at pH 7.5, 150 mM KoAc, 1 mM DTT, 0.1% NP-40).
		<ol>
			<li>Resuspend the magnetic beads in 1 mL of ice-cold K150 buffer.</li>
			<li>Incubate the magnetic beads for 2 min at 4 &#176;C with gentle rotation and pellet the magnetic beads with the help of a magnet. Remove and discard the supernatant.</li>
		</ol>
		</li>
		<li>Add the magnetic beads to the cell lysate supernatant from step 1.3.3 and incubate at 4 &#176;C for 2 h.</li>
		<li>Wash the protein bound magnetic beads 3x with K150 buffer, then 2x with K150 containing 250 mM NaCl, then 3x with K150 buffer as 1.3.5.</li>
		<li>Resuspend the beads in 300 &#956;L of FLAG elution buffer (100 mM NaCl, 20 mM Tris-HCl at pH 7.5, 5 mM MgCl2, 10% glycerol), add FLAG peptide to a final concentration of 0.5 &#956;g/&#956;L, and incubate with beads on a rotator at 4 &#176;C for 1 h, then pellet the magnetic beads with the help of a magnet.</li>
		<li>Collect the supernatant which contains the eluted MOV10 proteins, determine the concentration, and store at -80 &#176;C for future use.</li>
	</ol>
	</li>
</ol>
2. Nucleic acid preparation
<ol>
	<li>Purchase DNA and RNA oligonucleotides (oligos) with or without biotin labels from a suitable source. Dilute each oligo in RNase-free ddH2O to 20 &#956;M and keep it at -80 &#176;C for the future use. 
	NOTE: The oligo sequences of DNA/RNA substrates used in this study are listed in Table 2.</li>
	<li>Prepare the following mixture for the double-stranded RNA (dsRNA) annealing reaction for MOV10 helicase activity assay: mix 60 mM N-2-hydroxyethylpiperazine-N-ethane-sulphonic acid (HEPES) at pH 7.5, 6 mM KCl, 0.2 mM MgCl2 and RNase-free ddH2O in a final reaction volume of 20 &#956;L.</li>
	<li>Anneal RNA oligos to form RNA duplex by heating a mixture of the biotin-labeled top strand (2 &#956;M, final concentration) and a 1.5-fold of its unlabeled complementary bottom strand in the annealing buffer (step 2.2) at 95 &#176;C for 5 min, and then slowly cool it to room temperature (RT).</li>
</ol>
3. In vitro biochemical assays
<ol>
	<li>EMSA and enzymatic reactions
	<ol>
		<li>For the MEIOB EMSA assay, mix 50 mM Tris HCl at pH 7.5, 2 mM MgCl2, 50 mM NaCl, 10 mM EDTA, 2 mM dithiothreitol (DTT), 0.01% NP-40, 0.8 mM (or other relevant concentrations as shown in Figure 2 and Figure 3) MEIOB protein, and 10 nM biotin-labeled oligonucleotides and ddH2O in a final reaction volume of 20 &#956;L. Incubate at RT for 30 min, and add 5x stop buffer (125 mM EDTA, 50% glycerol) to stop the reaction. &#160;</li>
		<li>Set up MEIOB nuclease activity reactions as described in step 3.1.1 but without the addition of 10 mM EDTA.</li>
		<li>For the MOV10 helicase activity assay, mix 50 mM Tris-HCl at pH 7.5, 20 mM KoAc, 2 mM MgCl2, 0.01% NP-40, 1 mM DTT, 2 U/&#956;L RNase inhibitor, 10 nM biotin-labeled RNA substrate, 2 mM adenosine triphosphate (ATP), 100 nM RNA trap, and 20 ng of MOV10 protein and ddH2O in a final reaction volume of 20 &#956;L. Incubate the reaction mixture at 37 &#176;C for 10 min, 30 min, and 60 min. Add the 5x stop buffer to stop the reaction. 
		NOTE: RNA trap, a biotin-unlabeled oligo with sequence, complementarity to the labeled oligo, which prevents the unwound dsRNA from annealing again.</li>
	</ol>
	</li>
	<li>Polyacrylamide gels
	<ol>
		<li>Wash the gel plates (16 cm x 16 cm) and 1.5 mm combs. Assemble the gel electrophoresis units.</li>
		<li>To prepare a 10% native polyacrylamide gel, mix 14 mL of ddH2O, 1.25 mL of 10x Tris-boric acid-EDTA (TBE), 8.3 mL of 30% acrylamide, 1.25 mL of 50% glycerol, 187.5 &#956;L of 10% freshly prepared ammonium persulfate (APS), and 12.5&#160;&#956;L of tetramethylethylenediamine (TEMED).</li>
		<li>To prepare a 20% native polyacrylamide gel, mix 5.5 mL of ddH2O, 1.25 mL of 10x TBE, 1.25 mL of 50% glycerol, 16.7 mL of 30% acrylamide, 187.5 &#956;L of 10% APS, and 12.5 &#956;L of TEMED.</li>
		<li>Pour the acrylamide solution immediately to the gel and insert the comb. Let the mixture polymerize for approximately 30 min.</li>
	</ol>
	</li>
	<li>Gel running
	<ol>
		<li>Remove the comb, fill the tanks with the electrophoresis running buffer (0.5x TBE).</li>
		<li>Rinse the sample wells with 0.5x TBE buffer, then pre-run the gel at 100 V on ice for 30 min. Replace the running buffer with fresh 0.5x TBE.</li>
		<li>Load 20-25 &#956;L samples into each well.</li>
		<li>Use a 10% native acrylamide gel for the EMSA assay and a 20% native acrylamide gel for the enzymatic assays. Run electrophoresis at 100 V on the ice bath until the bromophenol blue marker has migrated to the bottom quarter of the gel.&#160;</li>
	</ol>
	</li>
	<li>Disassemble the gel plates, trim the gel by removing loading wells and unused lanes. Place the gel in 0.5x TBE buffer.</li>
	<li>Cut the filter paper and the nylon membrane to the size of the gel. Pre-wet the clean filter paper and the nylon membrane.</li>
	<li>Assemble the stack for transfer.
	<ol>
		<li>Place the pre-wet membrane onto the pre-wet filter paper.</li>
		<li>Place the gel on the membrane.</li>
		<li>Cover the gel with another layer of pre-wet filter paper.</li>
		<li>Remove all air bubbles by rolling a clean pipette from center to edge.</li>
	</ol>
	</li>
	<li>Transfer the samples from the gel to the membrane in a semi-dry electrophoretic apparatus at 90 mA for 20 min.</li>
	<li>Stop the transfer, and then dry the membrane on a new filter paper for 1 min.</li>
	<li>Crosslink the samples by irradiating the membrane at 120 mJ/cm2 for 45-60 s in a UV-light crosslinker equipped with 254 nm bulbs (auto crosslink function). Air dry the membrane at RT for 30 min.</li>
	<li>Chemiluminescence detection
	<ol>
		<li>Protocol 1: Use a standard volume of commercial chemiluminescent nucleic acid detection kit.
		<ol>
			<li>Add 20 mL of blocking buffer to the membrane and incubate for 15-30 min with gentle shaking on a rotator at 20-25 rpm.</li>
			<li>Prepare conjugate/blocking buffer solution by adding 66.7 &#956;L stabilized streptavidin-horseradish peroxidase conjugate to 20 mL of blocking buffer.</li>
			<li>Gently remove the blocking buffer and replace it with conjugate/blocking buffer. Incubate for 15 min on a rotator at 20-25 rpm.</li>
			<li>Wash the membrane 4x with shaking at 40-45 rpm for 5 min each.</li>
			<li>Add 30 mL of substrate equilibration buffer to the membrane. Incubate the membrane for 5 min with shaking at 20-25 rpm.</li>
			<li>Prepare substrate working solution by adding 6 mL of luminol/enhancer solution to 6&#160;mL of stable peroxide solution. Avoid light.&#160;</li>
			<li>Cover the entire surface of the membrane with substrate working solution and incubate for 5 min.</li>
			<li>Scan the membrane in a chemiluminescent imaging system for 1-3 s.</li>
		</ol>
		</li>
		<li>Protocol 2: Use 2x diluted commercial chemiluminescent nucleic acid detection kit and follow the steps 3.10.1.1-3.10.1.8.</li>
		<li>Protocol 3: Use self-made reagents6.
		<ol>
			<li>Prepare blocking buffer: mix 0.1 M Tris-HCl at pH 7.5, 0.1 M NaCl, 2 mM MgCl2, and 3% bovine serum albumin Fraction V. AP 7.5 buffer: mix 0.1 M Tris-HCl at pH 7.5, 0.1 M NaCl, and 2 mM MgCl2. AP 9.5 buffer: mix 0.1 M Tris-HCl at pH 9.5, 0.1 M NaCl, and 50 mM MgCl2. TE buffer: mix 10 mM Tris-HCl at pH 8.0, 1 mM EDTA at pH 8.0.</li>
			<li>Soak the membrane in blocking buffer at 30 &#176;C for 1 h.</li>
			<li>Add 8.5 &#956;L of streptavidin alkaline phosphatase to 10 mL of AP 7.5 Buffer. Shake the membrane gently in this solution at RT for 10 min. Then, wash the membrane 2x in 15 mL of AP 7.5 buffer for 10 min.</li>
			<li>Wash the membrane one more time in 20 mL of AP 9.5 buffer for 10 min. Add 20 mL of TE buffer to stop the reaction.</li>
			<li>Add 7.5 mL of development solution onto the membrane, and scan the membrane in a chemiluminescent imaging system for 1-3 s.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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H., 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=MOV10+Is+a+5'+to+3'+RNA+helicase+contributing+to+UPF1+mRNA+target+degradation+by+translocation+along+3'+UTRs.">MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.</a> Molecular Cell. 54 (4), (2014).</li><li>Talwar, T., 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=The+DEAD-box+protein+DDX43+(HAGE)+is+a+dual+RNA-DNA+helicase+and+has+a+K-homology+domain+required+for+full+nucleic+acid+unwinding+activity.">The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity.</a> The Journal of Biological Chemistry. 292 (25), 10429-10443 (2017).</li><li>Nagy, N. M., Konya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+fast+and+slow+consecutive+processes+by+heterogeneous+isotope+exchange+using+P-32+radiotracer.">Study of fast and slow consecutive processes by heterogeneous isotope exchange using P-32 radiotracer.</a> Journal of Radioanalytical And Nuclear Chemistry. 318 (3), 2349-2353 (2018).</li><li>Wilson, D. L., Beharry, A. A., Srivastava, A., O'Connor, T. R., Kool, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Probes+for+ALKBH2+Allow+the+Measurement+of+DNA+Alkylation+Repair+and+Drug+Resistance+Responses.">Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses.</a> Angewandte Chemie. 57 (39), 12896-12900 (2018).</li><li>Wilchek, M., Bayer, E. A., Livnah, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+biorecognition:+the+(strept)avidin-biotin+system+as+a+model+for+protein-protein+and+protein-ligand+interaction.">Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.</a> Immunology Letters. 103 (1), 27-32 (2006).</li><li>Trippier, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+strategies+for+the+biotinylation+of+bioactive+small+molecules.">Synthetic strategies for the biotinylation of bioactive small molecules.</a> ChemMedChem. 8 (2), 190-203 (2013).</li><li>Rodgers, J. T., Patel, P., Hennes, J. L., Bolognia, S. L., Mascotti, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biotin-labeled+nucleic+acids+for+protein+purification+and+agarose-based+chemiluminescent+electromobility+shift+assays.">Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.</a> Analytical Biochemistry. 277 (2), 254-259 (2000).</li><li>Panda, A. C., Martindale, J. L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Pulldown+of+Biotinylated+RNA+for+Detection+of+Protein-RNA+Complexes.">Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.</a> Bio-Protocol. 6 (24), (2016).</li><li>Bednarek, 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=mRNAs+biotinylated+within+the+5'+cap+and+protected+against+decapping:+new+tools+to+capture+RNA+-+protein+complexes.">mRNAs biotinylated within the 5' cap and protected against decapping: new tools to capture RNA - protein complexes.</a> Philosophical Transactions Of the Royal Society B-Biological Sciences. 373 (1762), (2018).</li><li>Souquet, B., 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=MEIOB+Targets+Single-Strand+DNA+and+Is+Necessary+for+Meiotic+Recombination.">MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination.</a> Plos Genetics. 9 (9), (2013).</li><li>Luo, 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=MEIOB+exhibits+single-stranded+DNA-binding+and+exonuclease+activities+and+is+essential+for+meiotic+recombination.">MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination.</a> Nature Communications. 4, 2788 (2013).</li><li>Xu, Y., Greenberg, R. A., Schonbrunn, E., Wang, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis-specific+proteins+MEIOB+and+SPATA22+cooperatively+associate+with+the+single-stranded+DNA-binding+replication+protein+A+complex+and+DNA+double-strand+breaks.">Meiosis-specific proteins MEIOB and SPATA22 cooperatively associate with the single-stranded DNA-binding replication protein A complex and DNA double-strand breaks.</a> Biology of Reproduction. 96 (5), 1096-1104 (2017).</li></ol>

No conflicts of interest are declared.

Investigating protein-nucleic acid interactions is critical to our understanding of molecular mechanisms underlying diverse biological processes. For example, MEIOB is a testis-specific protein essential for meiosis and fertility in mammals25,26,27. MEIOB contains an OB domain that binds to single-stranded DNA and exhibits 3&#39;&#160;to 5&#39;&#160;exonuclease activity26, which directly relates to its ph...

Protein-nucleic acid interactions are involved in many essential cellular processes such as DNA repair, replication, transcription, RNA processing, and translation. Protein interactions with specific DNA sequences within the chromatin are required for the tight control of gene expression at the transcriptional level1. Precise posttranscriptional regulation of numerous coding and noncoding RNAs necessitates extensive and complicated interactions between any protein and RNA2. These layers of gene expression regulatory mechanism comprise a cascade of dynamic intermolecular events, which are coordinated by interactions of transcription/epigenetic factors or RNA-binding proteins with their nucleic acid targets, as well as protein-protein interactions. To dissect whether proteins in vivo are directly or indirectly associated with nucleic acids and how such associations occur and culminate, in vitro biochemical assays are conducted to examine the binding affinity or enzymatic activity of proteins of interest on designed substrates of DNA and/or RNA.
Many techniques have been developed to detect and characterize nucleic acid-protein complexes, including the electrophoretic mobility shift assay (EMSA), also termed gel retardation assay or band shift assay3,4,5. EMSA is a versatile and sensitive biochemical method that is widely used for studying the direct binding of proteins with nucleic acids. EMSA relies on gel electrophoretic shift in bands, which are routinely visualized using chemiluminescence to detect biotin labels6,7, the fluorescence of fluorophore labels8,9, or by autoradiography of radioactive 32P labels10,11. Other purposes of biochemical studies are the identification and characterization of nucleic acid-processing activity of proteins, such as &#160;nuclease-based reactions to assess the cleavage products from nucleic acid substrates12,13,14 and DNA/RNA structure-unwinding assays to evaluate helicase activities15,16,17.
In such enzymatic activity assays, the radioisotope-labeled or fluorophore-labeled nucleic acids are often used as substrates due to their high sensitivity. Analysis of radiographs of enzymatic reactions involving 32P labeled radiotracers has been found to be sensitive and reproducible18. Yet, in an increasing number of laboratories in the world, the usage of radioisotopes is restricted or even prohibited due to the health risks associated with potential exposure. In addition to biosafety concerns, other drawbacks are the required necessary equipment to conduct work with radioisotopes, required radioactivity license, short half-life of 32P (about 14 days), and gradual deterioration of the probe quality due to radiolysis. Thus, alternative non-isotopic methods have been developed (i.e., labeling the probe with fluorophores enables detection by fluorescent imaging19). However, a high-resolution imaging system is needed when using fluorescently labeled probes. Biotin, a commonly used label, readily binds to biological macromolecules such as proteins and nucleic acids. Biotin-streptavidin system operates efficiently and improves detection sensitivity without increasing non-specific background20,21. Besides EMSA, biotin is widely used for protein purification and RNA pull-down, among others22,23,24.
This protocol successfully uses biotin-labeled nucleic acids as substrates for in vitro biochemical assays that include EMSA, in addition to enzymatic reactions in which biotin has not been commonly used. The MEIOB OB domain is constructed and two mutants (truncation and point mutation) are expressed as GST fusion proteins25,26,27, as well as mouse MOV10 recombinant FLAG fusion protein16. This report highlights the effectiveness of this combined protocol for protein purification and biotin-labeled assays for miscellaneous experimental purposes.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Germany</td><td>5242R</td><td></td></tr><tr><td>Chemiluminescent Imaging System</td><td>Tanon, China</td><td>5200</td><td></td></tr><tr><td>Digital sonifer</td><td>Branson, USA</td><td>BBV12081048A</td><td>450 Watts; 50/60 HZ</td></tr><tr><td>Semi-dry electrophoretic blotter</td><td>Hoefer, USA</td><td>TE77XP</td><td></td></tr><tr><td>Tube Revolver&amp;nbsp;</td><td>Crystal, USA</td><td>3406051</td><td></td></tr><tr><td>UV-light cross-linker</td><td>UVP, USA</td><td>CL-1000</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter&amp;nbsp;</td><td>Milipore, USA</td><td>UFC801096</td><td>4 ml/10 K</td></tr><tr><td>Nylon membrane</td><td>Thermo Scientific, USA</td><td>TG263940A</td><td></td></tr><tr><td>TC-treated Culture Dish</td><td>Corning, USA</td><td>430167</td><td>100 mm&amp;nbsp;</td></tr><tr><td>TC-treated Culture Dish</td><td>Corning, USA</td><td>430597</td><td>150 mm&amp;nbsp;</td></tr><tr><td>Microtubes tubes</td><td>AXYGEN, USA</td><td>MCT-150-C</td><td>1.5 mL&amp;nbsp;</td></tr><tr><td>Tubes</td><td>Corning, USA</td><td>430791</td><td>15 mL</td></tr><tr><td>&lt;strong&gt;Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ampicillin</td><td>SunShine Bio, China</td><td>8h288h28</td><td></td></tr><tr><td>Anti-FLAG M2 magnetic beads</td><td>Sigma, USA</td><td>M8823</td><td></td></tr><tr><td>ATP</td><td>Thermo Scientific, USA</td><td>591136</td><td></td></tr><tr><td>BCIP/NBT Alkaline Phosphatase Color Development Kit</td><td>Beyotime, China</td><td>C3206</td><td></td></tr><tr><td>CelLytic&lt;sup&gt;TM&lt;/sup&gt; M Cell Lysis Reagent&amp;nbsp;</td><td>Sigma, USA</td><td>107M4071V</td><td></td></tr><tr><td>ClonExpress II one step cloning kit&amp;nbsp;</td><td>Vazyme, China</td><td>C112</td><td></td></tr><tr><td>Chemiluminescent Nucleic Acid Detection Kit</td><td>Thermo Scientific, USA</td><td>T1269950</td><td></td></tr><tr><td>dNTP</td><td>Sigma-Aldrich, USA</td><td>DNTP100-1KT</td><td></td></tr><tr><td>DMEM</td><td>Gibco, USA</td><td>10569044</td><td></td></tr><tr><td>DPBS buffer</td><td>Gibco, USA</td><td>14190-136</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen, USA</td><td>AM9260G</td><td>0.5 M</td></tr><tr><td>EDTA free protease inhibitor cocktail</td><td>Roche, USA</td><td>04693132001</td><td></td></tr><tr><td>EndoFree Maxi Plasmid Kit&amp;nbsp;</td><td>Vazyme, China</td><td>&amp;nbsp;&lt;br/&gt; DC202</td><td></td></tr><tr><td>FastPure Gel DNA Extraction Mini Kit</td><td>Vazyme, China</td><td>DC301-01</td><td></td></tr><tr><td>FBS</td><td>Gibco, USA</td><td>10437028</td><td></td></tr><tr><td>FLAG peptide</td><td>Sigma, USA</td><td>F4799</td><td></td></tr><tr><td>Glycerol</td><td>Sigma, USA</td><td>SHBK3676</td><td></td></tr><tr><td>GST Bulk Kit</td><td>GE Healthcare, USA</td><td>27-4570-01</td><td></td></tr><tr><td>HEPES buffer</td><td>Sigma, USA</td><td>SLBZ2837</td><td>1 M&amp;nbsp;</td></tr><tr><td>IPTG</td><td>Thermo Scientific, USA</td><td>34060</td><td></td></tr><tr><td>KoAc</td><td>Sangon Biotech, China</td><td>127-08-02</td><td></td></tr><tr><td>Lipofectamin 3000 Transfection Reagent</td><td>Thermo Scientific, USA</td><td>L3000001</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Invitrogen, USA</td><td>AM9530G</td><td>1 M</td></tr><tr><td>NaCl</td><td>Invitrogen, USA</td><td>AM9759&lt;br/&gt; &amp;nbsp;</td><td>5 M&amp;nbsp;</td></tr><tr><td>NP-40</td><td>Amresco, USA</td><td>M158-500ML</td><td></td></tr><tr><td>Opti-MEM medium</td><td>Gibco, USA</td><td>31985062</td><td></td></tr><tr><td>PBS</td><td>Gibco, USA</td><td>10010023</td><td>PH 7.4</td></tr><tr><td>RNase Inhibitor</td><td>Promega, USA</td><td>N251B</td><td></td></tr><tr><td>Streptavidin alkaline phosphatase</td><td>Promega, USA</td><td>V5591</td><td></td></tr><tr><td>TBE</td><td>Invitrogen, USA</td><td>15581044</td><td></td></tr><tr><td>Tris-HCI Buffer&amp;nbsp;</td><td>Invitrogen, USA</td><td>15567027</td><td>1 M, PH 7.4</td></tr><tr><td>Tris-HCI Buffer&amp;nbsp;</td><td>Invitrogen, USA</td><td>15568025</td><td>1 M, PH 8.0</td></tr><tr><td>Tween-20</td><td>Sangon Biotech, China</td><td>A600560</td><td></td></tr></tbody></table>

in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

The protein structure of MEIOB and the expression constructs used in this study are illustrated in Figure 1A. OB folds in MEIOB are compact barrel-like structures that can recognize and interact with single-stranded nucleic acids. One of the OB domains (aa 136-307, construct A) binds single stranded DNA (ssDNA), the truncated protein (aa 136-178 truncations, construct C) and the point mutant form (R235A mutation, construct E) of MEIOB do not have DNA-binding activity<sup cla...

Watch this Scientific Journal Video about In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions at JoVE.com

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

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Research

JoVE Journal

Genetics

12.5K Views.  Nanjing Medical University. Dit protocol biedt een biotine-label platform voor de studie van eiwit-nucleïnezuur interacties die robuust, betrouwbaar, efficiënt en betaalbaar blijkt te zijn. Dezelfde partij van de biotine-gelabelde nucleïnezuren kan worden gebruikt over een lange periode van tijd, het behoud van reproduceerbaarheid van experimenten. Ten slotte kunnen alle hier beschreven tests met het biotinelabel binnen een dag worden uitgevoerd en vereisen ze geen speciale apparatuur.Het aantonen van de proce...

Video: In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Chemistry

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Bioengineering

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

Biochemistry

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Biology

Quantitative Analysis of RPA-ssDNA Binding Kinetics Using Label-Free Methods

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Neuroscience

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

Immunology and Infection

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

Cell-surface Biotinylation Assay

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or &#8220;recycled&#8221; back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively ...

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Cell Biology

Helicase Activity Measurement of a Target Protein Using Biotin-Labeled RNA Duplexes

Source: Yu, L. et al., <a href="https://app.jove.com/v/59830">In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the procedure to determine the helicase activity of a target protein to unwind the biotin-labeled dsRNA substrate. The activity of the enzyme was identified by analyzing the electrophoretic mobility shift, followed by a chemiluminescence assay using chemiluminescent enzyme-conjugated streptavidin.

Source: Yu, L. et al., In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions. J. Vis. Exp. (2019)
In this video, we ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

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