BIOL110L-Cell Biology Lab Spring Quarter 2012 Module 3-4 Wednesday May 30, 2012

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1 BIOL110L-Cell Biology Lab Spring Quarter 2012 Module 3-4 Wednesday May 30, 2012 PART I: Isolation of over-expressed GFP-Karyopherins from yeast extracts by affinity capture on nucleoporins Summary: The goal of the proposed experiment is to isolate GFP-karyopherins directly from yeast extracts using the FG domains of Nups as baits in affinity capture and BEAD HALO experiments. We will monitor the binding of endogenous Kaps, including the GFP-Kap, to Nups using SDS-PAGE and Coommassie blue staining (Affinity capture). We will visualize by microscopy Kap-GFPS bound to different Nup coated beads (Bead Halo), each representing a potential stepping stone for karyopherin transport across the interior of the NPC. We will interpret the results in relation to the Forest model of NPC conduit architecture. Preparative work you have already done: 1) Prepared 50 ul aliquots of yeast cytosol from a yeast strain overexpressing a GFP-Kap (module 3-1). The measured protein concentration of its cytosol was mg/ml. 2) Prepared ~1 ml of yeast cytosol from a wild type strain over-expressing GFP (module 3-3). The measured protein concentration of this cytosol was mg/ml. 3) Prepared a cell extract from E. coli strains over-expressing a GST-fusion with a Nup FG domain (module 3-2) and titrated the abundance of the GST-fusion in the extracts by capture on glutathione coated beads and comparison against a BSA standard (after SDS-PAGE and Coommassie blue staining). Knowing the abundance, you should now estimate the amount of E. coli extract needed to capture 10 ug of the GST-fusion on beads; this is for the affinity-capture experiment. The amount is ul (Modules 3-2 and 3-3). Likewise, estimate the amount of E. coli extract needed to capture 30 ug of the GST-fusion on beads; this is for the BEAD HALO experiment. The amount is ul. Once this data is collected, we will decide which FG domain(s) will be used by all in the affinity capture experiments (not all FG domains are appropriate for this step, why?). For the bead halo assay and the 18-well slides, we will need 0.25 to 0.50 ul of packed beads per well and 25 ul of yeast HSS fraction. Thus, we will need ~10 ul of beads with ~3 ug/ul concentration of the GST-Nup captured on them; hence, the 30 ug calculated for the beads used in BEAD HALO assays. Preparative work done for you by the TAs: Prepare eight 8% polyacrylamide gels to visualize the captured proteins by Coommassie blue staining. Bring all HSS fractions from wild type strains overproducing GFP or GFP Kaps. Equipment needed: Vacuum aspirators (4-6), microcentrifuges at 4 C & room temperature, heat blocks at 90 C, end-overend rotator (s) in the cold room, SDS-PAGE gel apparatus, Dewar with liquid nitrogen; ice buckets; metals blocks to serve as tube holders. Reagents needed: Bring 18-well slides suitable for BEAD HALO microscopy. Page 1

2 Glutathione coated beads (5 x 20 ul of 50% slurry per group in binding buffer) (keep extra beads just in case) Phenyl sepharose beads (1 x 20 ul of 50% slurry per group in binding buffer) (keep extra beads just in case) Binding buffer (20 mm Hepes ph 6.8, 150 mm KOAc, 2 mm MgOAc 2, 2 mm DTT, 0.1% Tween- 20). Need ~40 ml per group in 50 ml blue tubes (bring extra). Binding buffer supplemented with high magnesium chloride (20 mm Hepes ph 6.8, 150 mm KOAc, 300 mm MgCl 2, 2 mm DTT, 0.1% Tween-20) (250 ul per group) 6x Laemmli sample buffer (0.3 M Tris ph 6.8, 36% glycerol, 10% SDS, 0.012% bromophenol blue) with beta-mercaptoethanol (1:50). We will need 100 ul per group. 2x Laemmli sample buffer with beta-mercaptoethanol (1:50). Do not chill SDS buffer or it will solidify. We will need ~1 ml per group. Molecular weight markers (~2 ug or 0.4 ul in 50 ul 2x Laemmli sample buffer with BME (1:50) (one per group). Gel loading syringe (disposable) with 23G needle (8) Gel running buffer (25 mm Tris, 200 mm glycine, 0.1% SDS). Need ml per group. Gel fixing solution (10% acetic acid, 25% isopropanol) (8 x 50 ml = 400 ml) Gel staining solution (0.006% Coommassie blue; 10% acetic acid) (8 x 50 ml = 400 ml) Gel destaining solution (10% acetic acid) (8 x 3 x 50 ml = 1.2 liter) AFFINITY CAPTURE AND BEAD HALO ASSAYS: Logistics: For the affinity capture and BEAD HALO assays, we will first need to isolate and immobilize GST-Nup FG domain fusion proteins on glutathione-coated Sepharose beads to use as baits in the subsequent capture of yeast karyopherins: this loading of beads with the GST-nups is Stage I. The GST-Nups currently are in the crude bacterial MSS extracts you prepared (Module 3-2). You will then mix the nup-coated beads with a yeast HSS extract containing soluble proteins of yeast including endogenous karyopherins and the GFP-karyopherins. Only proteins (such as the Kaps) that bind with high specificity to the immobilized Nups (the baits) will be captured onto the bead surface during this phase or Stage II. For the affinity capture experiment we will elute the captured proteins with magnesium chloride (salt) and will separate them by size using SDS-PAGE. For the bead halo experiment, we will visualize the interactions directly using a fluorescence microscope. Stage I: Binding of GST-Nups to beads Note: One student will take care of Tubes A, B, C, D to be used in the affinity capture experiment; these tubes will have a TA designated GST-nup selected among those produced in Module 3-2. Note: The second student will takes care of Tube E to be used in the BEAD HALO assay; this tube will have the GST-Nup you prepared on Module ) Get an ice bucket and a metal block, and keep all tubes and buffers on the ice block (except the Laemmli sample buffers). Label four microcentrifuge tubes as Tubes A, B, C, D & E (TA will hand out); these contain a 20-ul of a 50% slurry of glutathione-coated beads (~10-ul of packed beads). 2) Thaw only 1 ml of each E. coli extract containing the GST-Nup assigned to your group and centrifuge at 4 C full-speed in a microcentrifuge for 5 min to remove insoluble aggregates. Transfer the supernatant to new tubes labeled with the GST-Nup used. Using the gel data from Module 3-3, calculate (a) how many microliters of extract you need to capture 10-ug of your GST-nup for Tubes A, B and C; these have the same GST-Nup. Then calculate how many microliters of extract you need to capture 10-ug of the assigned GST-nup for tube D (Nsp1 K>R mutant or phenyl sepharose). Finally, calculate how many microliters of extract you need to capture 30-ug of your GST-nup for Tube E. Page 2

3 For Tubes A, B and C we will need ul. For Tube D we will need ul. For Tube E we will need ul. 3) To Tubes A, B, and C add the calculated µl of extract needed to capture 10 ug of GST fusion. To Tube E add enough extract to capture 30 ug of the GST-fusion. Finally, to tube D add the calculated µl of extract needed to capture 10 ug of the GST-Nsp1 K>R fusion. Adjust the total volume of each tube to ~1 ml by adding binding buffer. Close well. 4) Incubate the bead slurry for 10 min tumbling end-over-end in the cold room to allow beads to capture the GST-nup. 5) Washing beads after binding: Sediment the beads in a microcentrifuge at 2,000 rpm for 30 sec, carefully vacuum-aspirate (with a gel loading tip) most of the supernatant, but don t get too close to the beads. 6) Perform the following three times: Add 1 ml of binding buffer to each tube, mix gently, sediment as before and aspirate off the supernatant. After three such washes, the beads are ready to bind proteins from yeast extracts. There should be ~10-ul of packed beads left in each tube. Tubes A, B, C and D go on to Stage II below (one student take over this part) Tube E, skip Stage II and proceed to the BEAD HALO section at the end (second student) Stage II: Binding of yeast karyopherins to immobilized GST-Nups on beads (tubes A, B, C and D) 1) Thaw three tubes of yeast cytosol, two containing the over-expressed Kap-GFP (~1 ml) from Module 3-1, and the other the over-expressed GFP (~1 ml) from Module 3-3. Vortex each tube gently and subject each to centrifugation at full-speed in the microcentrifuge for 3 min to remove debris. 2) In the meantime, add 1 ml of binding buffer to Tube A only (your negative control). When the centrifugation is done, dispense ~1 ml of the GFP over-expression cytosol into Tube B and 1 ml of the GFP-Kap cytosol into Tube C and Tube D. The total volume in each tube will be ~1 ml. Close the lids and incubate the tubes tumbling end over end for 15 min in the cold room. 3) Washing the beads after binding: Sediment the beads in Tubes A, B, C and D as before (2,000 rpm for 30 sec), and carefully vacuum-aspirate most of the supernatant, but don t get too close to the beads. Then perform the following step three times: [Add 1 ml of binding buffer, sediment as before, and aspirate most of the supernatant]. After the last wash, carefully remove the remaining buffer from the top of the beads with a gel-loading tip on a P200 pipetman. 4) To Tubes A, B, C and D, add 50-ul of Binding buffer containing 300 mm MgCl 2, mix in well by gently vortexing and incubate in your ice bucket for 3 min, mixing every ~30s by very gentle vortexing. Then sediment the beads as before and carefully collect 40-ul of each of the supernatants into new microcentrifuge tubes labeled A-sup, B-sup, C-sup and D-sup. Then re-label the remaining tubes with beads as A-pell, B-pell, C-pell or D-pell. SDS-PAGE (keep the tubes at room temperature from here on): 1) Add 50 ul of 2x Laemmli buffer to the tubes with beads (A-pell, B-pell, C-pell, D-pell) and vortex gently. Then add 15 ul of 6x Laemmli sample buffer to each of the supernatant tubes (A-sup, B-sup, C-sup, D-sup). Page 3

4 2) Heat all of the sup and pellet samples and the MW markers in a hot block for 8 min at 90 C. Then remove them, place them in the microcentrifuge and spin briefly for 10 sec. Then vortex each gently; they are ready to load in the gel. 3) While your samples are heating, prepare the gel. Remove the gel comb and the excess acrylamide, and assemble the gel into the electrophoresis unit. Top off wells with running buffer. 4) When ready to load the samples, with a syringe needle, carefully remove the buffer from each well. Then carefully load 30-ul of each sample (as indicated below) into the corresponding lanes starting from the left, in order 1 to 10. To deliver the sample, touch the gel-loading pipetman tip to the left side of the well touching the acrylamide divider and slowly deliver the solution. Use different tips for each sample. Do not mix the liquids from each well and make sure the liquid is falling in-between the plates. Lane 1: Lane 2: Lane 3: Lane 4 Lane 5 Lane 6 Lane 7: Lane 8: Lane 9: Lane ul of molecular weight markers 30 ul of Tube A sup (control for release of bait and any E. coli contaminant ) 30 ul of Tube B sup (contains captured proteins = endogenous Kaps only; no GFP) 30 ul of Tube C sup (contains captured proteins = endogenous Kaps and GFP-Kap) 30 ul of Tube D sup (contains captured proteins = endogenous Kaps and GFP-Kap) 30 ul of Tube A pellet (contains the bait) 30 ul of Tube B pellet (contains the bait, captured Kaps, no GFP) 30 ul of Tube C pellet (contain the bait, some captured Kaps, and the Kap-GFP) 30 ul of Tube D pellet (contain the bait, some captured Kaps, and the Kap-GFP)? 30 ul of 2 x Laemmli sample buffer (a blank ) 5) When all of the samples are loaded, overlay each (very gently!) with gel running buffer, then (very carefully) fill the back-chamber with running buffer until the buffer covers the wells. Use a 10 ml pipette, directing the stream AWAY from the wells. 6) Place the lid and run the gel at 20 milliamps constant current (voltage all the way up). If running two gels, run at 40 milliamps. 7) When the dye-front reaches the bottom of the gel (~60 min), stop the current, discard the running buffer, disassemble the gel apparatus, and carefully remove the top glass plate. Cut the very bottom right corner of gel to indicate orientation and remove excess acrylamide from wells. 8) Very carefully transfer the gel to the gel fix solution already in a tray. Grab the gel by the bottom edges at the corners. Use just enough fix solution to cover the gel. Fix for 15 min (the blue dye will disappear). 9) Discard the gel fix in the indicated hazards containers, rinse the gel twice with water (be careful when pouring off the liquid gently press the gel to the tray with a finger on a bottom corner to prevent it slipping out. 10) Add gel-staining solution, just enough solution to cover the gel. Stain for more than 10 min. The protein bands will become visible in a few minutes. Look in a light box. Page 4

5 11) When the gels are stained, pour off the stain in the indicated hazards container, and add geldestain solution. Shake in platform for ~5 min, decant in the indicated hazards container, and repeat until the gel background is clear and the proteins remain blue (~twice is enough). 12) Digitally scan the gel for documentation (the TAs will provide you with the images). Look for a new protein band appearing in the tube C sup and/or pellet) The expected molecular weights of the GFP-Kaps are: Protein MW (daltons) MW + GFP (daltons) GFP 29,371 Mtr2 20,784 50,155 Kap60 60,441 89,812 Mex67 67,351 96,722 Sxm1 108, ,774 Cse1 109, ,726 Kap , ,291 Msn5 142, ,487 *Look for your GFP-Kap among the captured proteins by comparing lanes 4 to lane 3; and lane 8 to lane 7. Lanes 2 and 6 are your negative control; they contain proteins from the E. coli extract, mostly the GST-Nup and its degradation products. PART II: BEAD HALO assay: 1) Thaw 1 ml of the HSS extract containing the over-produced GFP-Kap prepared by your group. Mix well, and aliquot 40-ul into 10 labeled tubes (i.e. label it Kap or follow code). Bring those to the front they will be redistributed. 2) To beads in Tube E, add 90 ul of binding buffer, and place on ice. 3) To each redistributed tube containing separate GFP-Kaps, add 10-ul of bead-slurry containing your GST-Nup (from Tube E). Remember, it is very important that you mix the slurry very well immediately before each withdrawal to guarantee equal distribution of beads. Use cut tips (for larger boars). 4) According to TA instructions and template, dispense 10-ul of each BEAD HALO mix into the designated wells in a 18-well microscope slide (avoid introducing bubbles!). Do not plunge pipet to the second stop. 5) Take pictures of the BEADS using the Rexach Lab microscope to visualize the binding of GFP-Kaps to Nup-coated beads. *Interpret the binding pattern in relation to the models of NPC conduit architecture. Page 5