Paul.xu at 15:28, 28 June 2018

2018-06-28T15:28:42Z

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Library recovery and expansion 1. Place frozen aliquots of yeast nanobody library (NbLib) at 30 °C to thaw, such that recoverable yeast exceed library diversity at least five-fold. For the full naïve library, a total of 2.5×109 viable cells are required.

2. Recover yeast in 1 L of ‘Yglc4.5 –Trp’, shaking at 230 RPM, 30 °C, overnight. You may want to take a small sample of the library cells immediately after resuspension and plate on YPD agar to measure the diversity.

3. Expand to 3 L of medium and allow yeast to grow to stationary phase. Approximately 48 hours, OD is usually between 10 and 30.

4. Measure OD600 of yeast to calculate density (OD600 of 1 ≈ 1.5 × 107 yeast)

5. Spin down cultures at 3500×g for 5 minutes and resuspend in ‘Yglc4.5 –Trp’ supplemented with 10% DMSO, such that final density is 1010 cells per mL. Aliquot to 2 mL cryovials and freeze in a cell freezing chamber at -80°C (Thermofisher Scientific cat# 5100-0001). Each vial should contain enough viable cells upon recovery to reconstitute a full library.

Induction of nanobody expression

Nanobody expression is under the control of the GAL1 promoter, so nanobodies are only produced on the cell surface when the yeast are grown in galactose-containing medium.

1. Induce nanobody expression of NbLib by dilution of ≥10 x library diversity into – Trp +galactose medium followed by shaking for 48 hours, 25 °C, 220 rpm. Use at least 5 × 109 yeast cells for the inoculum to ensure no clones are lost in passaging.

(We find best expression levels to be between 48 and 72 hours.) Note about sterility: It is essential to maintain a sterile environment throughout your selection. Unlike standard yeast work, your yeast cells will be passaged multiple times over the course of weeks, increasing the possibility of contamination. We recommend sterile filter tips and freshly sterilized media for every selection. Wipe down your hands, bench, and pipettes with ethanol often during the selection process. A sterile hood could be used as well, but usually a regular lab bench is fine so long as reasonable care is taken.

Nanobody expression test

Before any selection it’s a good idea to confirm that nanobody expression was effectively induced. We check this with a quick analytical flow cytometry assay as described below.

1. Measure cell density of induced NbLib yeast (1 OD600 ≈ 1.5 x 107 cells/mL) 2. Add 500 μl of ‘selection buffer’ to two microcentrifuge tubes 3. Pipette approximately 1×106 induced yeast into each microcentrifuge tube and centrifuge for 1 min at 3500 × g, 4 °C 4. Aspirate supernatant and resuspend pellet in 100 μl selection buffer 5. To one tube add ~0.5 μg of anti-HA antibody labeled with AlexaFluor647 or AlexaFluor488 (to check for the percentage of cells expressing nanobody) and 1 μM final concentration of your protein labeled with a spectroscopically distinct dye (to check if your antigen binds nonspecifically to yeast). Leave the other tube unstained as a control 6. Rock both tubes at 4 °C for 15 minutes 7. Spin down cells for 1 minute at 3500 × g, 4 °C 8. Aspirate supernatant and resuspend cells in 500 μL of selection buffer 9. Spin down cells as before and aspirate supernatant 10. Resuspend cells in 100 μl of selection buffer and assess nanobody expression level on flow cytometer using unstained sample to set gates

Note: The maximum number of expressing cells from the naïve library is ~25%. The typical number of expressing cells is ~15-20%. If expression is on the low end (~8-12%) consider increasing the number of cells used for nanobody selection to compensate. Example flow cytometer yeast gating:

Nanobody Selection The selection process below is at the heart of identifying clones that bind to your target protein. This protocol describes how to separate antigen binding nanobody clones from non-binders using magnetic cell sorting in two major steps: a “pre-clear” or negative selection step, followed by a positive selection step. These are usually done in series on the same day. The pre-clear step serves to deplete the library of any nanobodies that bind to things like secondary antibodies, magnetic beads, etc. The positive selection step isolates clones that bind to your antigen. After selection, you can expand your enriched cells and repeat the process until binders are highly enriched. 11. Spin down a suitable number of cells to have at least 10-fold over library diversity. For the naïve library (NbLib) you will need 5 × 109 cells. After one or more rounds of selection a smaller number of cells may be used based on estimated diversity. We recommend always using at least 1 × 107 cells, since smaller numbers are difficult to manipulate. Perform centrifugation for 3 minutes at 3500 × g, 4 °C 12. Aspirate media and resuspend yeast via pipetting in 10 mL of chilled selection buffer 13. Spin down yeast for 3 minutes at 3500 × g, 4 °C and aspirate supernatant 14. Resuspend yeast in 4.5 mL of selection buffer 15. Begin preclear to remove off-target binders from the naïve library by adding 500 μl of the ‘Miltenyi anti-fluorophore beads’ to the resuspended yeast (If using a secondary for selection, add that to the preclearing reaction as well. ~200 nM is typically sufficient to remove secondary binders) 16. Rock/Rotate slowly at 4 °C for 40 minutes 17. Approximately 20 minutes prior to the conclusion of incubation, place an ‘LD column’ on the Miltenyi MACS magnet. Put a 15 mL sterile falcon under the column and place a 50mL falcon cap over the top of the column to maintain sterility. Add 5 mL of selection buffer to the LD column to allow it to equilibrate before the next step. It will take ~20 minutes to flow through completely 18. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate buffer and unbound beads 19. Resuspend by pipetting in 5 mL of selection buffer. 20. Flow yeast over equilibrated LD column, collect flowthrough in new sterile 15mL centrifuge tube 21. Flush out remaining cells by washing column with an additional 2 mL of selection buffer 22. Remove LD column from magnet and discard 23. Spin down flow through at 3500 × g at 4 °C and aspirate supernatant 24. To resuspended yeast, add 5mL selection buffer containing fluorophore labeled antigen at a 1-1.5 μM concentration (if using a secondary to stain – preincubate the secondary with antigen before staining yeast and use a molar concentration of 2:3 secondary to antigen) 25. Rock or rotate slowly at 4 °C for 1 hour 26. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate supernatant 27. Resuspend yeast in 4.5 mL of selection buffer and 500 μl of anti-fluorophore beads 28. Rock or rotate slowly at 4 °C for 20 minutes 29. Approximately 5 minutes before end of incubation, place an ‘LS column’ on the Miltenyi MACS magnet. Put a 15 mL sterile falcon tube under the column and place a 50mL cap over the top of the column to maintain sterility. Keep plunger wrapped in package for later use. Add 5 mL of selection buffer to the LS column and allow it to equilibrate before the next step. It will take ~5 minutes to flow through completely 30. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate buffer and unbound beads 31. Resuspend yeast by pipetting in 3 mL of selection buffer and spin down as above 32. Aspirate supernatant and resuspend yeast in 3 mL of selection buffer 33. Remove 50 μl “pre-LS” aliquot of resuspended yeast and set aside for later analysis. Keep aliquot on ice. 34. Flow yeast over the equilibrated LS column and collect flow through in a new sterile 15 mL centrifuge tube 35. Wash column with 8 mL of selection buffer  36. Record approx. volume of flow through and remove 50 μl “flow through” aliquot for later analysis. 37. Remove LS column from magnet and place over fresh sterile 15 mL centrifuge tube 38. Add 5 mL of selection buffer to column and immediately use plunger to elute cells 39. Remove 50 μl “elution” aliquot from eluted cells and set aside for later analysis. Keep aliquot on ice. 40. Spin down elution at 3500 × g for 3 min at 4 °C and aspirate buffer taking care not to disturb the pellet 41. Resuspend yeast in 3 mL of –TRP +glucose in a 14 mL Falcon culture tube 42. Shake at 30 °C for about 24 hours to recover. 43. Use flow cytometry to compare the number of yeast from pre-LS and elution aliquots and use this ratio to get an estimate of the reduction in diversity of the library. Next steps: After recovering yeast, you can reinduce in galactose medium for another round of selection, and iterate until you isolate binders. It’s a good idea to use a different fluorophore for each selection round. We typically perform two MACS selections followed by a FACS sort to isolate binders. For a FACS sort, you can prepare your yeast by staining with antigen exactly as described above. The details of how to sort will depend on the specific project and sorting equipment being used. Once your selected library has a high proportion of active clones (usually 20% or more of the total cells), you can move on to screening. For screening, plate sorted yeast on –TRP plates for single clones. Grow single clones in a sterile deep-well 96-well plate, with one clone per well. Then induce expression and stain clones to identify those with the highest activity. Finally, a yeast miniprep or colony PCR can be used to isolate nanobody DNA for analysis and recombinant expression. Note that not every clone with activity on yeast retains activity in recombinant form. A large majority of clones will show activity in both formats, but there are some exceptions. Appendix 3.8 g of –Trp drop-out media supplement (US Biological) Yglc4.5 –Trp (1 liter) 6.7 g Yeast Nitrogen Base 10.4 g Sodium Citrate 7.4 g Citric Acid Monohydrate 10 mL Pen-Strep (10,000 units/mL stock) 20 g glucose Adjust pH to 4.5; Sterifilter (or sterifilter a stock of 20% glucose and add with antibiotic after autoclaving). 3.8 g –Trp drop-out media supplement (US Biological) –Trp (+glucose or +galactose - 1 liter) 6.7 g Yeast Nitrogen Base 10 mL Pen-Strep (10,000 units/mL stock) 20 g glucose or galactose (glucose for normal growth and galactose for induction of nanobodies) Adjust pH to 6; Sterifilter (or sterifilter a stock of 20% glucose or galactose and add with antibiotic after autoclaving). 20 g Bacto Peptone YPAD (1 liter) 20 g Glucose 10 g Yeast Extract 18 mg Adenine 10 mL Pen-Strep (10,000 units/mL stock) 20 mM HEPES pH 7.5 Selection buffer* 150 mM sodium chloride 0.1% (w/v) bovine serum albumin 5 mM maltose Sterile filter before use. *Many other buffers will work equally well, depending on your protein you may need to modify this. For GPCRs for instance, we usually add 0.1% (w/v) lauryl maltose neopentyl glycol detergent. No pH adjustment needed. Sterifilter or autoclave as appropriate MACS accessories catalog numbers Anti-Cy5/Anti-Alexa Fluor 647 MicroBeads Miltenyi Biotec Cat# 130-091-395 Anti-FITC MicroBeads Miltenyi Biotec Cat# 130-048-701 LD Columns Miltenyi Biotec Cat# 130-042-901 LS Columns Miltenyi Biotec Cat# 130-042-401

'''pYDS649HM'''

Library vector sequence: Nb.BV025 in pYDS649HM 8584 bp. Selectable in E. coli with ampicillin and in yeast by Trp dropout. Sequence with Gal1 promoter primer (“AATATACCTCTATACTTTAACGTC”).

GGTACCCGACAGGTTATCAGCAACAACACAGTCATATCCATTCTCAATTAGCTCTACCA CAGTGTGTGAACCAATGTATCCAGCACCACCTGTAACCAAAACAATTTTAGAAGTACTT TCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTG GATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTA TCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGC TATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGAC TCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGC GCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAG GAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAA CCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGA AGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTA ACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATC AACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGAT CGAATTCAACCCTCACTAAAGGGCGGCCGCC

Mating factor alpha leader

ATGAGATTCCCATCTATCTTCACCGCTG TTTTGTTCGCTGCTTCTTCTGCTTTGGCTGCTCCAGCTAACACCACCACCGAAGACGAA ACCGCTCAAATCCCAGCTGAAGCTGTTATCGACTACTCTGACTTGGAAGGTGACTTCGA CGCTGCTGCTTTGCCATTGTCTAACTCTACCAACAACGGTTTGTCTTCTACCAACACCAC CATCGCTTCTATCGCTGCTAAGGAAGAAGGTGTTCAATTGGACAAGAGAGAA

MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVIDYSDLEGDFDAAALPLSNSTNNGLSSTNTTIASIAAKEEGVQLDKRE

GCTAGC GCA

ASA

Nanobody coding sequence

CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTG CGCCTGAGCTGCGCGGCGAGCGGCTCTATTTCTCTGTACGCTGAAATGGGCTGGTATC GCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCGGTATTACTCGTGGTACTACTAC CTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAAC ACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCG CGGCTGACTACGTTCAGGGTAAATCTCGTGTTATCCCGCATTACTATTGGGGCCAGGG CACCCAGGTGACCGTGAGCAGC

QVQLQESGGGLVQAGGSLRLSCAASGSISLYAEMGWYRQAPGKERELVAGITRGTTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADYVQGKSRVIPHYYWGQGTQVTVSS

GGATCC

HA epitope tag

TACCCATACGATGTTCCAGATTACGCT

649 stalk sequence

CAA ACATCCTCACCCTCTACAGAAGCATCAGTATCAACCTCTAGTACCTCTTCCTCTGCTTCA CAATCATCTGACCCAACTACAACATCTTCGTCCAGTTCATCGTCTTCCCCATCTTCCCAA TCTGAGGAAATTTCATCATCACCGACGGTCTCAACTACACCATCAACTTCTTCATCATCT TCCTCAATGACTTCAACCACCACAACAAAGTCAATCTCAACTTCCACTACAAGTTCAGC TCCAGTTACAGATGTGACAGTTTCCTCATCGCCTAGTAAATCTACCTCTACTTCGACAA GTACAGAAACATCTAAAACACCTACTTCAATGACAGAGTATACATCTAGTACATCGATA ATTTCGACTCCAGTTAGTCACTCGCAGACAGGTTTGTCGGCTTCATCAAGTTCATCATC TACAACATCCGGTTCTTCGTCCACTAAATCAGAAAGTTCGACAACATCTGGCTCTTCCC AGTCCGTGGAATCAACCTCCAGCCACGCCACTGTTCTTGCTAATTCCGCAGAAATGGTC ACAACATCCTCTAGTTCATCCTCAACATCCGAAATGTCATTAACTAGTACTGCTACCAG TGTACCAGTCTCATCTAGTAGCAGTACGACATATTCTACTAGCGCATCTACACAAGCCG TCACTACAACATCTTCTTCCACTGTATCTACAACTTCTTCTAGTACAACGTTAACAAGCG CATTCACACATTCTTCAACCACATCGTCCGACCAGCCACCCAGCGACACTACAAGTCCA TCTACGACACACGAACCTCATGTAACCACTCAGACGTCATCAGAAACATCTTCTTCTAA GTCATCTTCTACTTCTTCTTCAAGTACATCTCAAACCTCTGAGTCTGCAACACCATCCGA TTCCGTATCACCTGGAAGTTCTACATCAACATCTTCTAGTAGCACTTCTACTTCCACTTC TATTTCCAGTGGAGAAACGACAACTTCTTCTTCTTCATCATCTGCCACGACCACTTCTAA CAGCGCAACCTTGTCAGTCTCTACCACACAAACTTCGATTGAAGCCAGTTCATCTACTA CATCTACATCTAGTTCAACAATTACAACTTCAAGTAGTAGCGCTCACATATCGTCGAAA TCTCAATCTAGTATTACCTATCCCTCTTCCTCGACATCTTCATCTACATCGTCCTCAATTT CTAGCGAATCTGAAAGTTTTGAATCGACATCAGCAGAAGATGCTCCATCAACAGCACC TTCATCAAGTGTCTCTTCTAAGAGTTCTACCTCTACAACATCAAGCACATCGACATCTTC AAGCACTCCATCTCCATCACCATCTTCCGTGAGTTCTTCCTCCACCAGCTCATTGACAAC TTCTGCTGTATCAACACCAGCTACCTCTCATTCTCAAAGTACTGTAGTAACCACCACTA CTATTACTACATCAACAGGTCCAGTGATGTCTACGACAACAGCTTATTCTTCTAGTTCT ACTAGCAGCTCGGAATCTTCTGAGGTTCAGTCTGTCATGTCATCTACGCCTAGTTCAAC ATCAACAACAACCAGTTCGGAATCTACTTCATCTAGCTCCACAGCTTCTACCTCACCAT CAACCTCGCAAACTTTCGAAACTTCTCCTACTATAGGAGGTGTCCCCTCAACCACTTCA TTTGTCTCTACGCCAACAACGAAATTGTCGCACACTACTTCCACTATGACAGCACAGTC CGATAGTAAGTCTACCCACTCCTCAAGCACATCGACAGAAGATAAATCATCCACTGCTT CTGCAGTTGACGAAAGCACTACAACATCCACTTCCACGGAGTCTACTACATCAGTAACA TCAGGCACCTCCCATTCCGCTAAAGAATCTTCGTCAAATTCTAAGGTGTATAGTTCACA GACAGCACACTCATCCATAAGTGTTGCATCATCACCTAGTACAAAG

GGCGCC

CAAATC CAATCTTCTATGGTTGAAATCTCTACCTACGCTGGTTCTGCTAACTCTGTTAACGCTGG TGCTGGTGCTGGTGCTTTGTTCTTGTTGTTGTCTTTGGCTATCATC

TAATGATTAATTAA CTCGAGATCTGATAACAACAGTGTAGATGTAACAAAATCGACTTTGTTCCCACTGTACT TTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGTTTTCCCAT GTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATATAGCTATT CACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATTTCAATT TGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGAATCGA ATCCTAAGAGAATTGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGC CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTG CAGCACATCCCCCCTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCC TTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCA TTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCC TAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTC GACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGA CGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAA ACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCC GATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA ACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG GTATTTCACACCGCAGGCAAGTGCACAAACAATACTTAAATAAATACTACTCAGTAATA ACCTATTTCTTAGCATTTTTGACGAAATTTGCTATTTTGTTAGAGTCTTTTACACCATTT GTCTCCACACCTCCGCTTACATCAACACCAATAACGCCATTTAATCTAAGCGCATCACC AACATTTTCTGGCGTCAGTCCACCAGCTAACATAAAATGTAAGCTTTCGGGGCTCTCTT GCCTTCCAACCCAGTCAGAAATCGAGTTCCAATCCAAAAGTTCACCTGTCCCACCTGCT TCTGAATCAAACAAGGGAATAAACGAATGAGGTTTCTGTGAAGCTGCACTGAGTAGTA TGTTGCAGTCTTTTGGAAATACGAGTCTTTTAATAACTGGCAAACCGAGGAACTCTTGG TATTCTTGCCACGACTCATCTCCATGCAGTTGGACGATATCAATGCCGTAATCATTGAC CAGAGCCAAAACATCCTCCTTAGGTTGATTACGAAACACGCCAACCAAGTATTTCGGA GTGCCTGAACTATTTTTATATGCTTTTACAAGACTTGAAATTTTCCTTGCAATAACCGG GTCAATTGTTCTCTTTCTATTGGGCACACATATAATACCCAGCAAGTCAGCATCGGAAT CTAGAGCACATTCTGCGGCCTCTGTGCTCTGCAAGCCGCAAACTTTCACCAATGGACCA GAACTACCTGTGAAATTAATAACAGACATACTCCAAGCTGCCTTTGTGTGCTTAATCAC GTATACTCACGTGCTCAATAGTCACCAATGCCCTCCCTCTTGGCCCTCTCCTTTTCTTTT TTCGACCGAATTAATTCTTAATCGGCAAAAAAAGAAAAGCTCCGGATCAAGATTGTAC GTAAGGTGACAAGCTATTTTTCAATAAAGAATATCTTCCACTACTGCCATCTGGCGTCA TAACTGCAAAGTACACATATATTACGATGCTGTCTATTAAATGCTTCCTATATTATATA TATAGTAATGTCGTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA AGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCC CGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT TTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTA TAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCTTGCCTGTAACTTACA CGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTT TTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAAT GAAGAAAAAAAAATAAACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATA TATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACATTCGATTAACGATAAGT AAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACAGACAAGATGAAACAA TTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGA TCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTT TTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTA TAGCACGTGATGAAAAGGACCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCT GATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGT CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCT GGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCA AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCA TAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTTCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGC GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGCAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC ATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGGAACGCCTGGTATC TTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG TCAGGGGGGCCGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCG GGCAGTGAGCGCAACGCAATTAATGTGAGTTACCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCCTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC ACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGGAATTAACCCTCACTAAAGGG AACAAAAGCTG
[[Category:Protocols]]

Yeast Display Nanobody - Revision history

Paul.xu at 15:28, 28 June 2018