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The authors thank members of the Barna Lab for helpful discussions.

1. mESC cell culture
NOTE: Prior to ALIBi activation and cell lysis, mESCs should be handled using standard sterile technique for cell culture. Centrifugation of intact mESCs should be performed at 200 &#215; g for 3 min at room temperature unless otherwise specified. Prewarm media to 37 &#176;C in a water bath before use unless otherwise specified. mESCs should be passaged into a fresh tissue culture plate that has been gelatinized overnight (see step 1.1).
<ol>
	<li>The day prior to thawing L31-FTA mESCs, coat the bottom of a 10 cm plate with 5 mL of 0.1% gelatin. Incubate at 37 &#176;C overnight.</li>
	<li>Combine mESC media components (Table 2) and pass through a 0.2 &#181;m filter. Store at 4 &#176;C for up to 1 month.</li>
	<li>On the day of thawing, warm a 5 mL aliquot of media for diluting the thawed cells and a 10 mL aliquot for plating them.</li>
	<li>Retrieve a cryovial containing ~5 &#215; 106 L31-FTA mESCs from liquid nitrogen storage. Immediately thaw by swirling the vial bottom in a 37 &#176;C water bath.</li>
	<li>Gently mix 0.5&#8211;1 mL of media from the 5 mL aliquot with the cryovial contents as the vial capacity allows, then transfer the cryovial contents back to the 5 mL media aliquot.</li>
	<li>Pellet the cells by centrifugation. Aspirate the supernatant.</li>
	<li>Resuspend the cell pellet with the 10 mL media aliquot.</li>
	<li>Aspirate the gelatin from the 10 cm plate and immediately transfer cell suspension into the plate. Incubate at 37 &#176;C.</li>
	<li>Every 24 h (except on passaging days), aspirate old media from the plate and replace with 10 mL of fresh media.</li>
	<li>When colonies are 70%&#8211;80% confluent (typically 48 h after plating), aspirate the old media and rinse twice with 10 mL of Dulbecco&#8217;s phosphate-buffered saline (DPBS).</li>
	<li>After aspirating the DPBS, coat the plate with 2 mL of 0.05% trypsin (Table 2). Incubate at 37 &#176;C for 5 min.</li>
	<li>Neutralize the trypsin by adding 4 mL of cold mESC media to the plate. Immediately use a 5 or 10 mL serological pipet to pipet up and down 15x in the plate to dissociate colonies into a single-cell suspension.</li>
	<li>Transfer the suspension into a conical tube. Optionally, remove an aliquot to count cells using a hemocytometer.</li>
	<li>Pellet the cells by centrifugation. Aspirate the supernatant.</li>
	<li>Resuspend the cell pellet in fresh media. Plate 5 &#215; 106cells (typically 1:6 passaging ratio) in a fresh gelatinized 10 cm plate and incubate at 37 &#176;C. Repeat steps 1.9&#8211;1.15 as needed until ready to proceed with transfection.</li>
	<li>To make frozen cell stocks, trypsinize a plate of mESCs (as in steps 1.10&#8211;1.14). Resuspend the cell pellet in freezing media (250&#8211;500 &#181;L per 5 &#215; 106 cells), aliquot into cryovials, and freeze slowly in a cell freezing container at -80 &#176;C overnight before transferring to liquid nitrogen storage.</li>
</ol>
2. Transfection
NOTE: Perform transfection on a day when mESCs are ready for passaging. Plasmids may begin expressing as early as 3-5 h after transfection. Once expressing, we recommend handling cells in the dark and minimizing exposure to temperatures below 37 &#176;C to prevent premature activation, since previous work has shown that Magnet optogenetic domains dimerize not only under blue light illumination but also upon exposure to cold31. Keep plates in a dedicated incubator or shield a shelf in a shared incubator from light with aluminum foil. When handling cells outside the incubator, turn off or block all room lights and windows beforehand, and use a red-light headlamp to see. Do not leave the incubator door open for longer than necessary. Take only small batches of plates out of the incubator at a time, and rest plates on an insulating surface (i.e., a clean styrofoam box lid) while handling to minimize contact with room-temperature work surfaces. Warm media fully before use. Do not inspect plates under a light-emitting microscope.
<ol>
	<li>The day before transfection, gelatinize a 10 cm plate.</li>
	<li>On the day of transfection, aspirate gelatin from the plate and add 7.5 mL of media to prewarm at 37 &#176;C. Prewarm reduced-serum medium at 37 &#176;C for resuspending cells.</li>
	<li>Dilute 18.75 &#181;L of the transfection reagent in 250 &#181;L of reduced-serum medium. Tap gently to mix and incubate for 5 min at room temperature.</li>
	<li>If co-expressing two plasmids encoding the N- and C-fragments, add 3.75 &#181;g of each plasmid to 250 &#181;L of reduced-serum medium. If only expressing one plasmid (i.e., a GFP negative control or a full-length BirA positive control), add 7.5 &#181;g of the single plasmid instead.</li>
	<li>Add the plasmid/reduced-serum medium mix to the transfection reagent/reduced-serum medium mix and tap/invert to mix. Incubate at room temperature for 15 min.</li>
	<li>In the meantime, trypsinize and pellet mESCs (steps 1.10&#8211;1.14). Resuspend the pellet in warmed reduced-serum medium to a concentration of 10 &#215; 106 cells/mL.</li>
	<li>Add 7.5 &#215; 106 (750 &#181;L) resuspended cells to the plasmid/transfection reagent/reduced-serum medium mixture. Invert the tube to mix. Incubate for 10 min at RT, inverting the tubes occasionally to keep cells suspended.</li>
	<li>Invert the tubes again briefly and add the mESC/plasmid/transfection reagent/reduced-serum medium mixture to the plate containing warmed media. Gently rock the plate to evenly distribute the cells.</li>
	<li>Incubate the plate at 37 &#176;C for 3&#8211;5 h, allowing the cells to adhere within this time. 
	NOTE: From this point on, we recommend observing the above precautions against exposure to light and temperatures below 37 &#176;C.</li>
	<li>Gently aspirate the old media. Add fresh warmed media, pipetting down the side of the plate to avoid dislodging cells. Resume incubation at 37 &#176;C.</li>
	<li>If activating/lysing at 24 h after transfection, proceed to section 3. If planning to activate/lyse at 48 h after transfection, change media ~24 h after plating the transfected cells.</li>
</ol>
3. ALIBi activation and cell lysis
NOTE: Continue to handle cells/samples in the dark until the beginning of elution (step 5.4). Once cells have been chilled on ice (step 3.15), keep samples cold and use only chilled reagents until the beginning of elution. Ensure that working surfaces, equipment, reagents, and plastics are RNAse-free by using freshly opened items and wiping or rinsing hard surfaces with an RNAse decontaminating solution and 70% ethanol. Wear gloves whenever handling samples or equipment. For samples intended for MS proteomics, also minimize keratin contamination by keeping the work space dust-free; cover bare skin, and tie back long hair.
<ol>
	<li>Prechill the centrifuge to 4 &#176;C.</li>
	<li>With lights off, move the transfected plates to a temporary 37 &#176;C incubator so they will not be exposed to temperature fluctuation in the main incubator after the transilluminator is placed inside.</li>
	<li>Wipe down the blue light transilluminator with 70% ethanol and connect it to power. Place the transilluminator in the main incubator and allow incubator conditions to re-equilibrate while continuing with the setup.</li>
	<li>Warm an aliquot of media to be dosed with biotin and cycloheximide (CHX) (10 mL of media per 10 cm plate) to 37 &#176;C. If running control samples without supplemental biotin, also warm a separate aliquot to be dosed with CHX only. 
	NOTE: Some buffer components can be mixed the day before use and stored overnight at 4 &#176;C (see Table 2). Note that different lysis and wash buffer recipes were used for experiments intended for western blot or MS proteomics and those intended for RNA sequencing.</li>
	<li>Chill an aliquot of DPBS to be dosed with CHX (20 mL per 10 cm plate).</li>
	<li>Prepare the RNAse removal buffer, salt wash, lysis buffer, and wash buffers (see Table 2). For buffers with dense/viscous components such as glycerol and Triton X-100, pipet the completed buffer to ensure even mixing. Chill buffers on ice prior to use. 
	NOTE: For bead washing steps, briefly spin the tube containing beads in their current buffer in a pocket centrifuge to collect the contents at the bottom. Place the tube in a magnetic rack for 1 min or until beads have fully migrated. Aspirate the supernatant without touching the bead pellet. Without letting the bead pellet dry, add the next buffer. To resuspend the bead pellet, either pipet gently or roll the closed tube between one&#8217;s fingers while holding it against the magnetic rack, such that the beads are quickly pulled in multiple directions by the magnet. Finally, invert the resuspended beads several times to ensure the inside walls of the tube are washed.</li>
	<li>Resuspend the stock bottle of streptavidin beads by gentle pipetting or rocking. Transfer 450 &#181;L of beads to a protein low-binding tube for each 10 cm plate of mESCs.</li>
	<li>Following the above NOTE on bead washing, discard the bead storage buffer and wash beads 2x with 900 &#181;L of RNAse removal buffer. 
	NOTE: Proceed to the next step without delay; extended time in RNAse removal buffer may decrease the beads&#8217; binding capacity.</li>
	<li>Wash beads 1x with 900 &#181;L of salt wash.</li>
	<li>Wash beads 2x with 900 &#181;L of Wash I Buffer. After resuspending beads in the second round of Wash I buffer, store the beads in the Wash I buffer at 4 &#176;C while performing activation and cell lysis.</li>
	<li>Add biotin (50 &#181;M final concentration; 1:1,000 dilution of 50 mM stock) and CHX (100 &#181;g/mL final concentration; 1:1,000 dilution of 100 mg/mL stock) as appropriate to the warmed aliquot(s) of media. Keep at 37 &#176;C until use.</li>
	<li>Add CHX (100 &#181;g/mL final concentration; 1:1,000 dilution of 100 mg/mL stock) to the cold aliquot of DPBS and keep chilled until use. 
	NOTE: The following two steps describe activation of ALIBi via supplemental biotin and blue light illumination. An ideal duration of activation is long enough to yield an adequate amount of ribosome biotinylation for downstream assays, but short enough to preserve spatiotemporal resolution. Ideal activation time may vary based on parameters such as the cell type, scale of the experiment, expression level of the split enzymes, abundance of ribosomes near the organelle of interest, and sensitivity of the analysis technique. We recommend 15 minutes as a starting point, which is the activation time used in our representative results (Figure 2) and in our accompanying work30.</li>
	<li>Aspirate media from the transfected plates. Immediately add 10 mL of the warm media containing CHX &#177; biotin per plate.</li>
	<li>For any negative control plates not to be illuminated, place them in a dark incubator. For plates to be illuminated, place them directly on top of the transilluminator, turn on the transilluminator, and incubate for 15 min.</li>
	<li>When illumination is complete, immediately wash the plates 2x with 10 mL of cold DPBS + CHX per plate. Place the plates on ice between and after the washes. After aspirating the second wash, tilt the plate and aspirate any residual DPBS.</li>
	<li>Keeping each plate as flat as possible on ice, proceed to lysis by adding 2 mL of ice-cold lysis buffer. Incubate plate on ice for 5 min, tapping plate to dislodge cells 2&#8211;3x during that time. 
	NOTE: if kept dark under foil and on ice, plates can be moved out of the cell culture area to another dark space during this time.</li>
	<li>Dislodge any remaining cells by using a micropipette to pipet the lysate up and down on the plate surface while avoiding formation of bubbles, then transfer lysate to a chilled and labeled protein low-binding tube. 
	NOTE: OPTIONAL: For some difficult-to-lyse organelles such as the nucleus and nucleolus, sonication may help.</li>
	<li>In the case of difficult-to-lyse organelles, sonicate all samples in the experiment for 45 s at 4 &#176;C (power setting of 3 watts). If the sonicator design requires insertion of a probe into an open sample tube, transfer the lysate initially from the plate into a larger tube to prevent overflow during sonication; then, transfer it to a 2 mL tube.</li>
	<li>Incubate the lysate on ice for 15 min.</li>
	<li>Centrifuge the lysate at 17,000 &#215; g for 3 min at 4 &#176;C. Transfer the supernatant (clarified lysate) to a chilled protein low-binding tube without disturbing the pellet of cell debris. 
	NOTE: If RNA-seq analysis is intended, an aliquot of the clarified lysate can now be collected as the total RNA fraction (see Supplemental File 1, &#8220;Recommendations for RNA sequencing&#8221; step 1.1) for details. 
	NOTE: If the cell quantities, transfection/culture conditions, and lysis conditions have been uniform for all samples, we typically assume lysate concentrations are comparable between samples and proceed without quantifying total protein concentration. This minimizes the elapsed time between activation and affinity purification.</li>
</ol>
4. Affinity purification
<ol>
	<li>Pellet the washed streptavidin beads using the magnetic rack. Aspirate the Wash I buffer.</li>
	<li>Transfer 1,200 &#181;L of clarified lysate to the tube containing the washed beads. Pipet gently to resuspend the beads. Store the remainder of the clarified lysate at -80 &#176;C.</li>
	<li>Incubate the lysate with beads at 4 &#176;C in the dark for 1 h on the tube rotator.</li>
	<li>During this incubation, prepare the TEV elution buffer (Table 2).</li>
</ol>
5. Elution
<ol>
	<li>Following the notes on bead washing in section 3, pellet the beads on a magnetic rack and transfer the supernatant to a chilled protein low-binding tube. Store the supernatant at -80 &#176;C.</li>
	<li>Wash beads 2x with 900 &#181;L of Wash I buffer.</li>
	<li>Wash beads 1x with 900 &#181;L of Wash II buffer. 
	NOTE: After completion of this step, samples no longer need to be handled in the dark.</li>
	<li>Add 450 &#181;L of TEV elution buffer to the beads.</li>
	<li>Incubate beads in TEV elution buffer for 1 h at room temperature on the tube rotator. For smaller-scale experiments using low volumes of TEV elution buffer, incubate the tubes on a shaker at 1,000&#8211;2,000 rpm instead.</li>
	<li>Pellet the beads on the magnetic rack. Transfer the eluate to a protein low-binding tube. If proteomics analysis or western blot is intended, store the eluate at -80 &#176;C. If RNA-seq analysis is intended, see Supplemental File 1 &#8220;Recommendations for RNA sequencing&#8221; step 1.2 for eluate storage instructions.</li>
	<li>OPTIONAL: To preliminarily determine whether the affinity purification has yielded appreciable rRNA/mRNA (indicating that ribosomes have been isolated), analyze eluates on a microvolume spectrophotometer using TEV elution buffer as a blank. 
	NOTE: Eluates from cells containing the activated system should have greater absorbance at 260 nm than eluates from cells lacking the split enzymes, blue light/biotin, or L31-FTA.</li>
</ol>
6. Western blot
NOTE: Until lysate and flow-through samples have been denatured in Laemmli buffer, we recommend continuing to avoid light exposure. We use precast gels, commercially available transfer buffer and running buffer, and a semi-dry transfer system (see the Table of Materials). SDS-PAGE and western blot are performed according to the manufacturers&#8217; instructions for these components, unless otherwise stated below.
<ol>
	<li>Add 6x Laemmli buffer to the lysate, flowthrough, and/or eluate samples to a final concentration of 1x Laemmli buffer. Heat at 95 &#176;C in a heat block or thermocycler for 10 min. If condensation forms on the tube lid/walls, spin down tubes briefly.</li>
	<li>Set up a gel with running buffer in the gel tank, per the tank manufacturer&#8217;s instructions. Load the samples and the ladder.</li>
	<li>Run the gel at 125 V for 45&#8211;75 min until the dye front approaches the bottom of the gel. In the meantime, prepare PBST and blocking buffers (Table 2).</li>
	<li>Perform a semi-dry transfer per the transfer system manufacturer&#8217;s instructions. 
	NOTE: For all membrane washing/incubation steps, ensure that the membrane is fully submerged and place the membrane tray on an orbital or tilting rocker. For a 10 x 15 cm tray, this requires 25&#8211;50 mL of buffer.</li>
	<li>Remove the membrane from the transfer stack and proceed to the next step immediately to ensure the membrane does not dry out. 
	NOTE: OPTIONAL: Dry spots on the membrane can result in uneven blocking or probe binding, especially with probes that have short incubations such as streptavidin-horseradish peroxidase. To avoid this issue, we re-wet membranes after transfer in 100% methanol for 30 s, then in water for 5 min. This measure is similar to general recommendations for re-wetting PVDF membranes that have dried after transfer32,33.</li>
	<li>Wash the membrane in PBST for 3 x 10 min at room temperature.</li>
	<li>Following the specified conditions in Table 3 for each western blot probe, perform the blocking, washing, probe incubation, and secondary antibody incubations.</li>
	<li>After the final wash in step 6.7, mix the ECL substrate components per the manufacturer&#8217;s instructions and coat the membrane evenly with it. Capture the image on a chemiluminescence gel imaging system.</li>
</ol>

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The authors have no conflicts of interest to declare.

ALIBi offers several advantages relative to existing techniques for studying subcellular translation and has potential future applications in ribosome biology and beyond. This protocol requires similar or less time compared to sequential lysis and centrifugation21,22 and is potentially applicable to any subcellular compartment that can be genetically targeted with a localizing peptide sequence. Compared to constitutively active BirA10...

There is an important need for tools that can isolate biological samples from specific subcellular compartments to better investigate how subcellular organization and trafficking factor into cellular processes. mRNA translation is a key example of such a process: growing evidence suggests that translation is highly compartmentalized and, in particular, that many mRNAs are localized to the organelles where their encoded proteins will reside1,2. However, less is known about the subcellular localization of translational machinery. Ribosomes begin their existence in the nucleolus and nucleus, where assembly from ribosomal proteins (RPs) and ribosomal RNAs (rRNAs) begins. They are then exported to the cytoplasm for the final stages of assembly after which they begin translating mRNAs3,4. Canonical sites of translation include the cytoplasm and endoplasmic reticulum; the latter is where integral membrane proteins and proteins destined for membrane-bound organelles or extracellular secretion are synthesized5. More recently identified sites of localized translation include the mitochondria, peroxisome, and neuronal processes6,7,8,9,10,11,12. Interestingly, evidence is also emerging that translational machinery features such as ribosome-associated proteins, post-translational modifications, rRNA modifications, and non-canonical stoichiometry of core ribosomal&#160;proteins are involved in regulatory mechanisms that govern translation of specific mRNAs and parameters such as translation fidelity and rate13,14,15,16,17,18,19,20. For example, hundreds of proteins have been identified that interact with the ribosome and could potentially lead to differences in subcellular ribosome composition13. However, techniques for isolating subcellular ribosome populations have been limited to date; thus, much remains unknown about how the composition of translational machinery contributes to regulating localized mRNA translation. The advent of versatile methods for isolating subcellular ribosome populations, particularly those compatible with mammalian model systems, will allow for exploration of these translation machinery features at the subcellular level.
The earliest methods for isolating subcellular fractions containing ribosomes were based on principles of sequential lysis and centrifugation21,22. Typically, these methods are limited to certain organelles and often require extensive processing times. A more recent method is based on the biotin ligase BirA, which specifically biotinylates an AviTag peptide substrate10,23,24. The BirA can be genetically localized to the organelle of interest in cells where ribosomes have been tagged with AviTag. However, because BirA is active at physiologic concentrations of biotin, it is necessary to culture cells under conditions of biotin depletion in order to avoid constitutive and non-specific biotinylation of AviTag-bearing ribosomes10,24. This limits the use of BirA in cell types that are sensitive to biotin depletion25. Finally, promiscuous biotinylation methods have been developed that use engineered enzymes such as BioID, TurboID, and APEX26,27,28. Upon induction, these enzymes produce a reactive biotin intermediate that covalently modifies any lysine residues within a radius of the enzyme. Promiscuous biotinylation methods are highly useful for mapping subcellular proteomes, but less informative for distinguishing proteins in the general vicinity of localized ribosomes as opposed to proteins that are in closer proximity with them.
Here, we demonstrate our recently developed method for labeling and isolating subcellular ribosome populations, called AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi). This method leverages an engineered split BirA fused to optogenetic enhanced Magnet (eMag) switches (Figure 1A) that heterodimerize under blue light illumination29; and a peptide tag consisting of FLAG epitope, tobacco etch virus (TEV) protease cleavage site, and AviTag (FTA) fused to the Rpl31 ribosomal protein (Figure 1B). The split enzyme can be genetically targeted to selected organelles, is inactive at physiologic concentrations of biotin, and can be reconstituted within minutes upon blue light illumination and the addition of supplemental biotin (Figure 1C-E). Subsequent cell lysis, affinity purification, and non-denaturing enzymatic elution via TEV protease cleavage yields ribosomes and co-purifying molecules such as mRNAs and ribosome-associated proteins, which are suitable for downstream analytic methods such as RNA sequencing and mass spectrometry proteomics (Figure 1A). Therefore, this method reveals not only ribosome composition but also the mRNAs to which ribosomes are bound. In a recent article describing the seminal application of ALIBi to characterize subcellular ribosome populations, we successfully targeted the split enzyme to the cytoplasm, nucleolus, nucleus, endoplasmic reticulum, plasma membrane, and mitochondria (Table 1)30. The resulting comparison of translational machinery between these subcellular compartments led to novel and unexpected findings regarding translation at the endoplasmic reticulum and mitochondria, as well as potential leads for future investigation of ribosome biogenesis factors in the nucleus and nucleolus.
This protocol contains a detailed demonstration of transfection and activation of the ALIBi system, cell lysis, affinity purification, and sample elution. We also discuss how to verify successful affinity purification via western blot and make recommendations for performing downstream RNA sequencing or mass spectrometry (MS) proteomic analysis (see Supplemental File 1). This protocol is written for experiments using mouse embryonic stem cells (mESCs) expressing Rpl31 fused to FTA (L31-FTA) homozygously from the endogenous Rpl31 locus; this cell line is available from the authors upon request. The strategy for generating this cell line is described in Supplemental File 1 and in Supplemental Figure S1 and Supplemental Figure S2. Plasmids encoding the split enzymes targeted to several organelles are available on Addgene (catalog numbers #235608 to 235643). The design principles for targeting the split enzymes to these organelles is described in Supplemental File 1. The protocol assumes that each sample originates from mESCs grown in a 10 cm plate. We have found that the 10 cm scale yields sufficient material for mass spectrometry proteomics, while the 6-well scale is sufficient for western blots or RNA sequencing.

<table><tbody><tr><td>-80 &amp;deg;C storage</td><td></td><td></td><td>For cell culture</td></tr><tr><td>0.1% gelatin in water</td><td>MilliporeSigma</td><td>ES-006-B</td><td>For cell culture</td></tr><tr><td>0.2 &amp;micro;m filter units</td><td>Fisher</td><td>04-977-158</td><td>For cell culture</td></tr><tr><td>0.5% trypsin-EDTA (10X)</td><td>ThermoFisher</td><td>15400-054</td><td>For cell culture</td></tr><tr><td>1 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ThermoFisher</td><td>AM9010</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>1 M Tris pH 7.5</td><td>ThermoFisher</td><td>15567027</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>1 M Tris pH 8</td><td>ThermoFisher</td><td>AM9855G</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>1.5 and 2 mL microcentrifuge tubes, autoclaved</td><td></td><td></td><td>For transfection</td></tr><tr><td>1.5, 2 mL, and 5 mL protein low-binding microcentrifuge tubes</td><td>Eppendorf</td><td>022431081, 022431102,&lt;br/&gt; 0030108302</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>10 cm tissue culture plates</td><td></td><td></td><td>For cell culture</td></tr><tr><td>10 M NaOH</td><td>MilliporeSigma</td><td>72068-100ML</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>10x Tris/glycine/SDS running buffer concentrate</td><td>Bio-Rad</td><td>1610772</td><td>For Western blot</td></tr><tr><td>37 &amp;deg;C tissue culture incubator x 2</td><td></td><td></td><td>For cell culture. One of these serves as the main incubator for cell culture and illumination of transfected cells. Temporary space is needed in a second incubator on the day of ALIBi activation to house transfected cells while setting up the transilluminator in the main incubator.</td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td>For cell culture</td></tr><tr><td>5 M NaCl</td><td>ThermoFisher</td><td>AM9010</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>50 mM biotin</td><td>ThermoFisher</td><td>B20656</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>6x Laemmli buffer</td><td>FIsher</td><td>50-196-784</td><td>For Western blot</td></tr><tr><td>70% ethanol</td><td></td><td></td><td>For disinfection</td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A955</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>AcTEV protease kit</td><td>ThermoFisher</td><td>12575015</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>ALIBi C-fragment plasmid</td><td>Addgene</td><td>#235615, 235636 &amp;ndash; 235641</td><td>Midi-prep scale is required for transfection</td></tr><tr><td>ALIBi N-fragment plasmid</td><td>Addgene</td><td>#235614, 235635, 235642, 235643</td><td>Midi-prep scale is required for transfection</td></tr><tr><td>Aluminum foil</td><td></td><td></td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>ammonium bicarbonate</td><td>MilliporeSigma</td><td>5.3305</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>anti-FLAG antibody</td><td>MilliporeSigma</td><td>F3165</td><td>For Western blot</td></tr><tr><td>anti-GAPDH antibody</td><td>Cell Signaling</td><td>5174</td><td>For Western blot</td></tr><tr><td>anti-mouse secondary antibody</td><td>GE Healthcare</td><td>NA931V</td><td>For Western blot</td></tr><tr><td>anti-rabbit secondary antibody</td><td>GE Healthcare</td><td>NA934V</td><td>For Western blot</td></tr><tr><td>anti-Rpl12 antibody</td><td>Abcam</td><td>ab175219</td><td>For Western blot</td></tr><tr><td>anti-Rpl31 antibody</td><td>Abcam</td><td>ab103991</td><td>For Western blot</td></tr><tr><td>anti-Rpl5 antibody</td><td>ProteinTech</td><td>15430-1-AP</td><td>For Western blot</td></tr><tr><td>anti-V5 antibody</td><td>ThermoFisher</td><td>R960-25</td><td>For Western blot</td></tr><tr><td>Apyrase</td><td>NEB</td><td>M0398L</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Beta-mercaptoethanol (55 mM)</td><td>ThermoFisher</td><td>21985-023</td><td>For cell culture</td></tr><tr><td>Blue light transilluminator</td><td>Clare Research Chemical</td><td>DR46B (small) or DR196 (large)</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher</td><td>BP1600-100</td><td>For Western blot</td></tr><tr><td>C18 tips (OMIX C18 tips)</td><td>Agilent</td><td>A57003100</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Cell freezing container</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Centrifuge fitted for 15 mL and/or 50 mL conical tubes</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Centrifuge for 1.5-2 mL microcentrifuge tubes</td><td></td><td></td><td>For lysis &amp;amp; affinity purification. Should be capable of refrigeration down to 4 C and speeds up to 17,000 g. Ideally, it is located in a room that can be temporarily darkened while transferring tubes in and out.</td></tr><tr><td>Clarity ECL kit</td><td>Bio-Rad</td><td>170-5061</td><td>For Western blot</td></tr><tr><td>Criterion gel tank</td><td>Bio-Rad</td><td>1656001</td><td>For Western blot</td></tr><tr><td>Criterion TGX pre-cast gels, 4-20%</td><td>Bio-Rad</td><td>5671095</td><td>For Western blot. Additional well sizes and layouts available</td></tr><tr><td>Cycloheximide</td><td>MilliporeSigma</td><td>C7698-5G</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Deoxycholate</td><td>MilliporeSigma</td><td>S1827</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Dithiothreitol</td><td>ThermoFisher</td><td>PIA39255</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>ThermoFisher</td><td>14190-250</td><td>For cell culture</td></tr><tr><td>ES-qualified FBS</td><td>MilliporeSigma</td><td>ES-009-B</td><td>For cell culture</td></tr><tr><td>Ethanol</td><td>Gold Shield</td><td>412828</td><td>For Western blot</td></tr><tr><td>Ethanol</td><td>MilliporeSigma</td><td>459828</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Formic acid</td><td>MilliporeSigma</td><td>A117</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Fragment size analyzer and consumables</td><td></td><td>Agilent</td><td>Optional: for RNA sequencing library preparation. Agilent 2100 Bioanalyzer</td></tr><tr><td>Freezing media</td><td>ThermoFisher</td><td>12648-010</td><td>For cell culture</td></tr><tr><td>Gel imaging system with chemiluminescence</td><td></td><td></td><td>For Western blot</td></tr><tr><td>Gel loading tips</td><td></td><td></td><td>For Western blot</td></tr><tr><td>Gel tank power source</td><td></td><td></td><td>For Western blot</td></tr><tr><td>Glycerol</td><td>MilliporeSigma</td><td>G5516-100ML</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>guanidinium-phenol reagent (TRIzol)</td><td>ThermoFisher</td><td>15596-018</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>guanidinium-phenol reagent for large sample volume (TRIzol LS)</td><td>ThermoFisher</td><td>10296010</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>guanidinium-phenol RNA extraction kit (DirectZol kit)</td><td>Zymo</td><td>R2060</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>HALT Protease Inhibitor</td><td>ThermoFisher</td><td>78425</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Hemocytometer</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Heptafluorobutyric acid</td><td>ThermoFisher</td><td>52411</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>High pH fractionation kit</td><td>ThermoFisher</td><td>84868</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Hydroxylamine</td><td>MilliporeSigma</td><td>467804</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>IGEPAL CA-630</td><td>MilliporeSigma</td><td>I8896</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>Implementation of guide RNA scoring algorithm</td><td>Benchling</td><td>https://www.benchling.com/</td><td>Optional: for selecting guide RNAs to insert the FTA sequence into an endogenous ribosomal protein locus. User-friendly interface that implements the guide RNA scoring algorithm from Doench, J.G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology. 34 (2), 184&amp;ndash;191, doi: 10.1038/nbt.3437 (2016).</td></tr><tr><td>Instant milk powder, nonfat</td><td>Safeway</td><td>116100001</td><td>For Western blot</td></tr><tr><td>Insulating surface, i.e. styrofoam box lid</td><td></td><td></td><td>For transfection</td></tr><tr><td>Iodoacetamide</td><td>ThermoFisher</td><td>A39271</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Knockout DMEM</td><td>ThermoFisher</td><td>10829-018</td><td>For cell culture</td></tr><tr><td>L-Glutamine (100x)</td><td>MilliporeSigma</td><td>TMS-002-C</td><td>For cell culture</td></tr><tr><td>L31-FTA mouse embryonic stem cells</td><td></td><td></td><td>Available on request from the Barna Lab, mbarna@stanford.edu</td></tr><tr><td>Light microscope with 4-10x magnification</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Lipofectamine 2000</td><td>ThermoFisher</td><td>11668-019</td><td>For transfection</td></tr><tr><td>Liquid nitrogen storage</td><td></td><td></td><td>For cell culture</td></tr><tr><td>LysC</td><td>Wako</td><td>125-05061</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Magnetic rack for 1.5-2 mL tubes</td><td>ThermoFisher</td><td>CS15000</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Methanol</td><td>VWR</td><td>EM-MX0475-1</td><td>For Western blot</td></tr><tr><td>Microcentrifuge tube rotator</td><td></td><td></td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Micropipettes and sterile or autoclaved micropipette tips</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Microseal for 96-well plates</td><td>Bio-Rad</td><td>MSB1001</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>Microvolume spectrophotometer</td><td>ThermoFisher</td><td>NanoDrop 2000</td><td>Optional</td></tr><tr><td>midi-prep kit (Plasmid Plus Midi Kit)</td><td>Qiagen</td><td>12945</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>mLIF (10,000 U/&amp;micro;L)</td><td>Gemini</td><td>400-495 10^7</td><td>For cell culture</td></tr><tr><td>Multiplex indices kit (NEBNext Multiplex Oligos for Illumina (Index Primers Sets 1 and 2))</td><td>NEB</td><td>E7335S, E7500S</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>MyOne C1 streptavidin Dynabeads</td><td>ThermoFisher</td><td>65002</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>No-weigh DTT</td><td>ThermoFisher</td><td>PIA39255</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Non-essential amino acids (100x)</td><td>MilliporeSigma</td><td>TMS-001-C</td><td>For cell culture</td></tr><tr><td>Non-expressing plasmid backbone (TOPO vector kit)</td><td>ThermoFisher</td><td>450031</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>Orbital or tilting rocker</td><td></td><td></td><td>For Western blot</td></tr><tr><td>PBS powder</td><td>Fisher</td><td>BP661-10L</td><td>For Western blot</td></tr><tr><td>pCAGEN</td><td>Addgene&amp;nbsp;</td><td>11160</td><td>Optional: for designing additional organelle-targeted ALIBi split enzyme constructsd</td></tr><tr><td>PCR thermocycler tubes</td><td></td><td></td><td>For Western blot</td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>ThermoFisher</td><td>15140163</td><td>For cell culture</td></tr><tr><td>pH strip</td><td>Fisher</td><td>8881-1</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Plastic trays for membrane incubations</td><td></td><td></td><td>For Western blot</td></tr><tr><td>Pocket centrifuge for 1.5-2 mL centrifuge tubes</td><td></td><td></td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Pocket centrifuge for PCR tubes</td><td></td><td></td><td>For Western blot</td></tr><tr><td>poly(A) mRNA isolation kit (NEBNext Poly(A) mRNA Magnetic Isolation Module)</td><td>NEB</td><td>E7490</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>Polymerase master mix suitable for genotyping (MyTaq Hot Start Red Mix)</td><td>BioLine</td><td>BIO-25048</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>Protein ladder</td><td>ThermoFisher</td><td>26617</td><td>For Western blot</td></tr><tr><td>protein precipitation kit (ProteoExtract kit)</td><td>MilliporeSigma</td><td>539180</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Proteinase K</td><td>MilliporeSigma</td><td>3115879001</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>Purified water</td><td></td><td></td><td>For Western blot. i.e. from Milli-Q (MilliporeSigma) filtration system</td></tr><tr><td>puromycin</td><td>MilliporeSigma</td><td>P8833</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>PX459</td><td>Addgene</td><td>62988</td><td>Optional: for inserting FTA into an endogenous ribosomal protein locus</td></tr><tr><td>Red-light headlamp</td><td></td><td></td><td>For transfection</td></tr><tr><td>Reduced-serum medium (OptiMEM)</td><td>ThermoFisher</td><td>11058021</td><td>For transfection</td></tr><tr><td>RNA column kit (RNA Clean &amp;amp; Concentrator-5 kit)</td><td>Zymo</td><td>R1016</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>RNA library prep kit (NEBNext Ultra II Directional RNA Library Prep kit)</td><td>NEB</td><td>E7760S</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>RNA non-stick 1.5 mL microcentrifuge tube</td><td>ThermoFisher</td><td>AM12450</td><td>Optional: for RNA sequencing library preparation</td></tr><tr><td>RNAseZap</td><td>ThermoFisher</td><td>AM9784</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Semi-dry transfer kit containing 5x transfer buffer, PVDF membrane, and blotting stacks</td><td>Bio-Rad</td><td>1704272</td><td>For Western blot</td></tr><tr><td>Semi-dry transfer system</td><td>Bio-Rad</td><td>1704150</td><td>For Western blot</td></tr><tr><td>Shaker for 1.5-2mL microcentrifuge tubes</td><td></td><td></td><td>For lysis &amp;amp; affinity purification. Should be capable of speeds up to 1000-2000 rpm</td></tr><tr><td>Sonicator (Microson Ultrasonic Cell Disruptor XL)</td><td>Misonix</td><td>XL-2000</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Sterile 15 mL and 50 mL conical tubes</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Sterile cryovials, 1.5-2 mL capacity</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Sterile serological pipettor and pipettes</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Streptavidin-HRP</td><td>ThermoFisher</td><td>N100</td><td>For Western blot</td></tr><tr><td>SUPERase RNAse Inhibitor</td><td>ThermoFisher</td><td>AM2696</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>SuperSignal West Femto Maximum Sensitivity Substrate kit</td><td>ThermoFisher</td><td>34095</td><td>For Western blot</td></tr><tr><td>Thermocycler or heat block</td><td></td><td></td><td>For Western blot</td></tr><tr><td>Thiourea</td><td>MilliporeSigma</td><td>T8656</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Tissue culture hood</td><td></td><td></td><td>For cell culture</td></tr><tr><td>Tissue culture space and bench space in room(s) that can be darkened</td><td></td><td></td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>TMT 6plex labels</td><td>ThermoFisher</td><td>90066</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>triethylammonium bicarbonate</td><td>ThermoFisher</td><td>T7408</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Triton X-100</td><td>MilliporeSigma</td><td>T8787-250ML</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Trypan Blue (0.4%)</td><td>Life Technologies</td><td>15250-061</td><td>For cell culture</td></tr><tr><td>Trypsin</td><td>ThermoFisher</td><td>90057</td><td>Optional: for mass spectrometry sample preparation</td></tr><tr><td>Turbo DNAse</td><td>ThermoFisher</td><td>AM2238</td><td>For lysis &amp;amp; affinity purification. Optional: for RNA sequencing library preparation</td></tr><tr><td>Tween-20</td><td>MilliporeSigma</td><td>P9416</td><td>For Western blot</td></tr><tr><td>Ultrapure water</td><td>ThermoFisher</td><td>10977023</td><td>For lysis &amp;amp; affinity purification</td></tr><tr><td>Urea</td><td>MilliporeSigma</td><td>U6504</td><td>Optional: for mass spectrometry sample preparation</td></tr></tbody></table>

isolation of subcellular ribosome subpopulations based on recombinant peptide tag-specific location-restricted illumination-enhanced biotinylation

Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation

Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of specific mRNAs is key to ensuring that proteins with compartment-specific functions are produced in the ideal milieu and in appropriate quantities. However, techniques for subcellular isolation and characterization of the ribosomes that translate these mRNAs have been limited to date, such that much remains unknown about how the composition of translational machinery contributes to regulation of localized mRNA translation.
Here, we demonstrate AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi), a method that combines epitope tagging, a newly developed optogenetically activated split-biotin ligase, and affinity purification to rapidly and specifically label and isolate ribosomes localized to any subcellular compartment of interest. First, CRISPR/Cas9 editing is used to fuse an AviTag peptide, a tobacco etch virus (TEV) protease cleavage site, and a FLAG epitope tag to a ribosomal protein. The split biotin ligase, fused to an organelle-targeting domain, is expressed in this cell line and is inactive under normal biotin concentrations. Upon activation by supplemental biotin and blue light illumination, the ligase biotinylates AviTagged ribosomes in the immediate vicinity, allowing for affinity purification of biotinylated ribosomes and associated proteins and mRNAs on streptavidin-coated beads. Non-denaturing elution via TEV protease cleavage yields samples suitable for downstream characterization of core ribosomal proteins, ribosome-associated proteins, and ribosome-bound mRNAs via RNA sequencing or mass spectrometry proteomics.
In this protocol, we review design principles for fusing AviTag to a ribosomal protein and targeting the split biotin ligase enzyme to the organelle of interest. We demonstrate activation of the ALIBi system, cell lysis, affinity purification, and sample elution. Finally, we discuss typical results and troubleshooting.

Western blot (protocol section 6) is the most expedient way to preliminarily assess the success of an experiment. When membranes are probed with streptavidin-HRP, lysate from cells where all components of the system were present should contain a band corresponding to biotinylated L31-FTA (Figure 2A, lane 20). The same band should be present, usually at greater intensity, if cells were transfected with full-length BirA as a positive control (Figure 2A, lanes 1–4)...

AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi) is a novel optogenetically activated method developed for labeling and affinity-purifying subcellular populations of ribosomes for downstream sequencing and proteomic analysis.

Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of ...

Our research investigates how ribosome composition and mRNA translation vary across subcellular domains to regulate local protein synthesis. Current methods lack spatial and temporal precision. ALIBi addresses this through optogenetically controlled proximity labeling of ribosomes.To begin, obtain L31FTA mouse embryonic stem cells and transfect them with ALIBi, GFP control, or full length BirA plasmids. After 24 to 48 hours of transfection, pre-chill the centrifuge to four degrees Celsius. With the lights off, move the transfected plates to a temporary 37-degree Celsius incubator.To prevent temperature fluctuations, once the transilluminator is placed in the main incubator, using 70%ethanol, wipe down the blue light transilluminator. After connecting the transilluminator to power, place it in the main incubator. Allow the incubator conditions to reequilibrate, while continuing the setup.Warm an aliquot of media to 37 degrees Celsius for dosing with biotin and cyclohexamide, or CHX. If preparing control samples without supplemental biotin, warm a separate aliquot for CHX-only dosing. Chill an aliquot of DPBS intended for CHX dosing.Prepare the RNase removal buffer, salt wash, lysis buffer, and wash buffers. Using a pipette, mix buffers containing dense or viscous components to ensure even mixing. Chill the prepared buffers on ice before use.Re-suspend the stock bottle of streptavidin beads by gentle pipetting or rocking. Transfer 450 microliters of beads into a protein low binding tube for each 10 centimeter plate of cells. Place the re-suspended beads on the magnetic rack for one minute, or until the beads have fully migrated.Aspirate and discard the bead storage buffer without touching the bead pellet. Wash the beads two times with 900 microliters of RNAs removal buffer. Wash the beads once with 900 microliters of salt wash.Wash the beads two times with 900 microliters of wash I buffer. Store the beads in the wash I buffer at four degrees Celsius while performing activation and cell lysis. Add biotin and CHX to the warmed aliquot of media and keep at 37 degrees Celsius until use.Add CHX to the cold DPBS aliquot and keep it chilled until use. Aspirate the media from the transfected plates. Immediately add 10 milliliters of warm media containing CHX with or without Biotin to each plate.Place negative control plates in a dark incubator. Place the remaining plates directly on top of the transilluminator. Turn on the transilluminator and incubate the plates for 15 minutes.Wash each plate two times with the 10 milliliters of cold DPBS containing CHX. Place the plates on ice between and after washes. After aspirating the second wash, tilt the plate and aspirate any residual DPBS.Keeping the plate flat on ice, add two milliliters of ice-cold lysis buffer and incubate the plate on ice for five minutes. Tap the plate two to three times during incubation to dislodge cells. Using a micropipette, pipette the lysate up and down across the plate surface to dislodge any remaining cells.Transfer the lysate to a chilled labeled protein low binding tube. In difficult to lyse organelles, sonicate all samples for 45 seconds at four degrees Celsius. And if required, transfer the lysate into a larger tube before sonication.Transfer the sonicated lysate into a two-milliliter tube. Incubate the lysate on ice for 15 minutes. Centrifuge the lysate at 17, 000 G for three minutes at four degrees Celsius.Transfer the supernatant into a chilled protein low binding tube without disturbing the pellet. Using a magnetic rack, pellet the washed strepped avident beads. Aspirate the wash one buffer.Transfer 1, 200 microliters of clarified lysate to the tube containing the washed beads. Pipette up and down gently to re-suspend the beads. Store the remaining clarified lysate at minus 80 degrees Celsius.Incubate the lysate with the beads at four degrees Celsius in the dark on a tube rotator for one hour. During the incubation, prepare the Tobacco Etch Virus, or TEV elution buffer. Pellet the beads on a magnetic rack.Transfer the supernatant to a chilled protein low binding tube. Store the supernatant at minus 80 degrees Celsius. Wash the beads two times with 900 microliters of wash I buffer and once with 900 microliters of wash II buffer.Add 450 microliters of TEV elution buffer to the beads. Incubate the beads in TEV elution buffer for one hour at room temperature on a tube rotator. Pellet the beads on the magnetic rack.Transfer the elute to a protein low binding tube. Store the eluate at minus 80 degrees Celsius if proteomics analysis or Western blot is intended. Cells containing all system components showed a L31-FTA band, which was stronger with full length burray and faint or absent when components were omitted.Endogenous biotinilated carboxylase proteins were detected in all lysates at 70 to 80 kilodaltons and 120 to 130 kilodaltons. ANTV5 probing confirmed expression of the split bore fragments. Biotinillated L31FTA decreased in the flow through relative to the lysate.No biotinillated L31-FTA was present in the eluate following TEV-mediated elution. Anti-flag probing showed a cleaved L31-FTA band at a lower molecular weight than intact L31-FTA ribosomal protein. S12 probing demonstrated the presence of the small ribosomal subunit in the eluate.Anti-V5 probing showed that proteins that do not bind the ribosome were absent from the eluate. When 2.5 fold more lysate was used with the same 450 microliters of streptavidin coated beads, a substantial amount of biotinilated L31-FTA remained in the flow through. Endogenous biotinilated proteins were partially depleted in the flow through.Endogenous biotinillated proteins were absent from the eluet, but were recovered after heating post-dilution beads in lambly buffer. Anti-flag probing detected cleaved L31-FTA at a lower molecular weight than intact L31-FTA. Anti-V5 probing showed that the split burray fragments were absent from the eluate.ALIBi enables the identification of ribosome-bound proteins and mRNAs in specific subcellular regions, revealing differences in translation regulation. The key challenge is maintaining specific proximity labeling through careful control of targeting construct expression and illumination conditions. Following this protocol, affinity purified ribosomes and associated mRNAs can be analyzed using quantitative proteomic and transcriptomic approaches.

isolation-subcellular-ribosome-subpopulations-based-on-recombinant

Research

JoVE Journal

Biology

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef’s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Peptide-based Identification of Functional Motifs and their Binding Partners

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Stanford School of Medicine.  AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi) is a novel optogenetically activated method developed for labeling and affinity-purifying subcellular populations of ribosomes for downstream sequencing and proteomic analysis.

Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation

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