Protocol Scfv Isolateion

Protocol Scfv Isolateion

High throughput isolation of scFv

Material

  1. Buffers
  2. Wash buffer = 50 mM sodium phosphate buffer pH8.0,NaCl 0.3M, 5mMimidazole)
  3. Elution buffer = 50 mM sodium phosphate buffer pH8.0, NaCl 0.3M and 250 mMimidazole
  4. Equilibration buffer x10 = 3M NaCl and 0.5M Tris pH 9
  5. Preparation of affinity Gel
  6. Thoroughly RS the affinity gel (Sigma P6611) with gentle inversion and aliquot the appropriate quantity for use (15µlper ml of SN= 1.5 ml per plate) into a 15 ml tube.
  7. Wash 1x with 10 mlnanopure water -Centrifuge 5 min at 3000 rpm and discard SN
  8. Wash 1x with 10 ml of wash buffer- Centrifuge 5 min at 3000 rpm and discard SN
  9. Resuspend the rinsed affinity gel in 5 ml/plate of wash buffer
  10. Plates and accessories
  11. To grow and induce yeast = 2ml U96Nunc 278752. Autoclave before each use and rinse with water/10% bleach after each use.
  12. To store the yeast at -80ºC = 0.3 ml U96 Becton Dickinson 35 1177.
  13. To incubate affinity gel with SN = Deepwell plate 2ml V96 Ependorf 951033502
  14. To filter = Multiscreen HTS MSGVN2250
  15. To elute = polypropylene 96 well plate cat number: 12565502 fisher scientific
  16. Accessories
  17. To pour reagents for distribution with multipipet = reservoirs B-0820-4 Bioexpress
  18. To seal the wells during incubation with affinity gel = Deepwell mats Ependorf 951030147. Autoclave before each use and rinse with water/10% bleach after each use.
  19. To align the filter plate with the reservoir plate during centrifugation = alignment frames Millipore MACF09604.

Methods

  1. Transfer yeast SN
  2. Transfer 100 µl of yeast culture from 2ml U96 to sterile 0.3ml U96 culture plate; centrifuge, discard SN and resuspend in 100 µl of sterile deionized water + 15% glycerol for long term storage at -80ºC
  3. Centrifuge 2ml U96 culture plate for 5min at 3000rpm
  4. Transfer 800 µl of culture SN (careful to NOT pipet the pellet!) in 2ml V96
  1. Incubate affinity gel with SN in 2ml x 96 well plates

-Use reservoirs to pour the buffer for distribution with multichannel pipet in 96 well plates -

  1. Distribute Equilibration Buffer in culture SN. Use a ratio of 1/10 (80 µl/well for 800 µlof SN)
  2. Distribute 50 µl/well of affinity gel (mix often!) in culture SN
  3. Seal the lids with a plastic mat (autoclaved Deepwell mat). Press firmly.
  4. Rotate 1 hour in the cold room
  5. Quick spin the plates with the mat lids on at 3000rpm for 3mins.
  1. Transfer the SN in filter plate
  2. Pre-wet wells of filter plate with 150 µL wash buffer and vacuum (max pressure =15).
  3. Transfer the SN onto the filter plate, 250µl by 250µl (use 300µl tips); vacuum blot after each 250µl. Prepare an empty box of tips to save tips in between 2 transfers.
  1. Wash affinity gel pellet
  2. Add 100 µL of wash bufferper well. Seal the plate with a plastic sticker.
  3. Ramp to 800-900 rpm on plate shaker for 30 seconds then reduce to 300 rpm for 10 minutes.
  4. Vacuum blot. Repeat 1 more time
  1. Elution x 2
  2. Add 55µlElution Buffer per well and seal with plasticsticker. Place a polypropylene 96 well plate + alignment frame under the filter plate. Wrap the two plates in foil to keep them together
  3. Ramp to 800-900 rpm on plate shaker for 30 seconds then reduce to 300 rpm for 15 minutes RT
  4. Centrifuge at 3000rpm for 5 minto elutescFvinto the polypropylene plate.
  5. Repeat elution 1 more time.
  1. Perform Elisa immediately or store up to 1 weekat 4oC.
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