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Preprint

KIF22 Immunofluorescence

JS Jason Stumpff

Last updated date: Oct 22, 2023 Views: 759 Forks: 0

original An abbreviated version of this protocol was published in eLife
Pathogenic mutations in the chromokinesin KIF22 disrupt anaphase chromosome segregation
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  • Cells fixed in 1% paraformaldehyde (PFA) in MeOH, 5 mL 16% PFA +  75 mL ice-cold MeOH.
  • Coverslips fixed on ice for 10 minutes.
  • Washed 3X in TBS for 5 min each, beaker changed each wash.
  • Coverslips transferred to parafilm in humidified chamber, cell side up.
  • Blocked in 20% goat serum in AbDil, 25 uL/coverslip, for 1 hr at room temp on shaker.
    • AbDil= TBS, pH 7.4; 1% BSA, 0.1% Triton X-100, 0.1% sodium azide
  • After block, cells washed 2X in 1X TBS for 5 min each in beakers
  • Primary antibodies for overnight incubation diluted AbDil:
    • GeneTex rabbit anti-KIF22 (GTX112357) 1:250
      • 2 uL anti-KIF22 +  498 uL AbDil
  • 25 uL 1º antibody applied to each coverslip, incubated overnight at 4ºC  on shaker
    • Time: __________
  • Coverslips removed from overnight primary incubation
    • Date: __________       Time: __________
  • Washed 2X in 1X TBS  for 5 min each
  • Primary antibodies for 1 hour incubation diluted in AbDil:
    • Mouse DM1a 1:500
      • 1 uL antibody + 499 uL AbDil
  • 25 uL 1º antibody applied to each coverslip, incubated 1 hour at room temp on shaker
  • Washed 2X in 1X TBS for 5 min each
  • Secondary antibodies diluted in AbDil:
    • All secondary antibodies diluted 1:500
    • Rabbit 594, Mouse 647 (GFP in 488)
      • 1 uL each + 498 uL AbDil
  • 25 uL 2º antibody applied to each coverslip, 1 hour at room temp on shaker, in dark
  • Washed 3X in 1X TBS for 5 min each
  • Coverslips mounted in Prolong Gold with DAPI, cured at room temp overnight in the the dark, then stored at 4ºC
     
Copyright: © 2023 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Stumpff, J(2023). KIF22 Immunofluorescence. Bio-protocol Preprint. bio-protocol.org/prep2480.
  2. Pathogenic mutations in the chromokinesin KIF22 disrupt anaphase chromosome segregation. eLife. DOI: 10.7554/eLife.78653

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