Difference between revisions of "20.109(S23):M2D6"

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(Introduction)
(Introduction)
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==Introduction==
 
==Introduction==
  
===Part 2: Perform antibody staining for γH2AX assay===
+
==Protocols==
 +
 
 +
 
 +
===Part 1: Perform antibody staining to determine Fet4 transporter expression===
 +
 
 +
'''Fix yeast and plate them on coverslips'''<br>
 +
#Obtain your 12-well plates  with coated coverslips from the front laboratory bench.
 +
#Obtain yeast cultures for your group:
 +
#*1 culture of W303&#945; yeast
 +
#*1 culture of W303&#945; yeast expressing Fet4
 +
#*1 culture of W303&#945; yeast expressing Fet4_mutant
 +
*Obtain a media blank at the front bench to check optical density of your cultures
 +
 
  
 
'''Complete primary staining steps'''<br>
 
'''Complete primary staining steps'''<br>
  
 
[[Image:IMG_5503.JPG|thumb|right|350px|'''Immunofluorescence staining chamber''']]
 
[[Image:IMG_5503.JPG|thumb|right|350px|'''Immunofluorescence staining chamber''']]
#Obtain your 12-well plates from the front laboratory bench.
+
#Obtain your 12-well plates with coated coverslips from the front laboratory bench.
 
#Gather an aliquot of 1 X TBS from the front laboratory bench.
 
#Gather an aliquot of 1 X TBS from the front laboratory bench.
 
#*Prepare 1.2 mL solution of 0.2% Triton X-100 (v/v) in 1X TBS in a micro centrifuge tube using the 10% Triton stock is at the front laboratory bench.
 
#*Prepare 1.2 mL solution of 0.2% Triton X-100 (v/v) in 1X TBS in a micro centrifuge tube using the 10% Triton stock is at the front laboratory bench.
Line 54: Line 66:
 
*Why is it important to wash the secondary antibody from the coverslip before imaging?
 
*Why is it important to wash the secondary antibody from the coverslip before imaging?
 
*What stain is used following secondary antibody?  What cellular component is stained in this step?  And why is this useful?
 
*What stain is used following secondary antibody?  What cellular component is stained in this step?  And why is this useful?
 +
 +
===Part 2: Set up experimental conditions for cytotoxicity assay===
 +
 +
==Reagents list==
 +
*4% paraformaldehyde (Electron Microscopy Sciences)
 +
*Sorbitol-citrate buffer: 1.2M sorbitol (Sigma), 10mM citric acid (Sigma), pH 7.0
 +
*Zymolase (Zymo Research)
 +
*permeabilization buffer: 0.1% Tween-20 in Phosphate buffer saline (PBS) (from Invitrogen)
 +
*blocking buffer: 1% bovine serum albumin (BSA) in PBS (BSA from Sigma)
 +
*1:500 Alexafluor-488 conjugated primary antibody to V5 tag, mouse (from Genescript)
 +
*1:1000 DAPI (from ThermoFisher)
 +
*Glycerol (Sigma)
 +
**Synthetic dropout-uracil (SD-U) media: 0.17% yeast nitrogen base without amino acid and ammonium sulfate (BD Bacto), 0.5% ammonium sulfate (Sigma), 0.192 % amino acid mix lacking uracil (Sigma), 2% glucose (BD Bacto), 0.1% adenine hemisulfate (Sigma)
 +
**Cadmium chloride (Sigma), [stock] = 100mM
  
 
==Navigation links==
 
==Navigation links==
 
Next day: [[20.109(S23):M2D7 |Confirm transporter expression and cell survival of yeast exposed to metal]] <br>
 
Next day: [[20.109(S23):M2D7 |Confirm transporter expression and cell survival of yeast exposed to metal]] <br>
 
Previous day: [[20.109(S23):M2D5 |Analyze ICP-OES data]] <br>
 
Previous day: [[20.109(S23):M2D5 |Analyze ICP-OES data]] <br>

Revision as of 18:55, 2 February 2023

20.109(S23): Laboratory Fundamentals of Biological Engineering

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Spring 2023 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Drug discovery        M2: Protein engineering        M3: Project design       

Introduction

Protocols

Part 1: Perform antibody staining to determine Fet4 transporter expression

Fix yeast and plate them on coverslips

  1. Obtain your 12-well plates with coated coverslips from the front laboratory bench.
  2. Obtain yeast cultures for your group:
    • 1 culture of W303α yeast
    • 1 culture of W303α yeast expressing Fet4
    • 1 culture of W303α yeast expressing Fet4_mutant
  • Obtain a media blank at the front bench to check optical density of your cultures


Complete primary staining steps

Immunofluorescence staining chamber
  1. Obtain your 12-well plates with coated coverslips from the front laboratory bench.
  2. Gather an aliquot of 1 X TBS from the front laboratory bench.
    • Prepare 1.2 mL solution of 0.2% Triton X-100 (v/v) in 1X TBS in a micro centrifuge tube using the 10% Triton stock is at the front laboratory bench.
    • Prepare 2.5 mL solution of 1% BSA (v/v) in 1X TBS in 15 mL conical tube. 10% BSA stock is at the front bench.
      • One of the preparations will be the blocking solution used in Step #8 and the other preparation will be used in Step #9 for the primary antibody solution.
  3. Obtain a staining chamber from the front bench and add a damp paper towel to each side of the parafilm.
    • Label the parafilm with experimental details.
  4. Obtain a fine gauge (26 3/8) needle and a pair of tweezers from the front laboratory bench.
    • Carefully press the tip of the needle against the benchtop to bend it into a right angle such that the beveled side of the needle is the interior angle.
  5. Use the 'hook' created with the needle to lift the coverslip from the bottom of the well, then use the tweezers to 'catch' the coverslip.
    • Practice plates with coverslips will be available at the front laboratory bench.
  6. When you are confident with your ability to retrieve the coverslips from the wells, move one coverslip from each condition from your 12-well plates to the staining chamber. Cell-side UP!
    • The cell-side of the coverslip is the side that was facing up in the well of the 12-well plate.
  7. Quickly permeabilize the cells by adding 150 μL of the 0.2% Triton X-100/TBS solution to each coverslip and incubate for 10 min at room temperature.
  8. Aspirate the 0.2% Triton X-100/TBS solution and add 150 μL of BSA blocking solution to each coverslip, then incubate for 60 min at room temperature.
  9. With 15 min remaining of the blocking solution incubation, prepare the primary antibody.
    • Dilute the mouse anti-γH2AX antibody 1:1000 in the 1.2 mL aliquot of BSA blocking solution.
  10. Aspirate the block solution and add 150 μL of the diluted primary antibody solution to each coverslip before moving the next. Do not let the coverslips dry!
  11. Cover your staining chamber with the lid to incubate at room temperature.
  12. Incubate samples in the primary antibody solution for ~1 h.

Complete secondary staining steps

  1. Aspirate the primary antibody and wash each coverslip by pipetting 200uL of TBS to the top of the coverslip, then use pipet or aspirator to remove liquid.
    • Complete a total of 3 washes. At the final wash leave the liquid on the coverslip.
  2. Retrieve aliquot of diluted secondary antibody, Alexa Fluor 488 goat anti-mouse (diluted 1:200 in blocking solution) from front bench.
  3. Aspirate the wash liquid from one coverslip and immediately add 150 μL of the diluted secondary antibody to the coverslip.
    • Complete this step for each coverslip individually as it is important that the coverslips do not dry!
  4. Cover your coverslips to protect them from light.
  5. Incubate samples at 4 °C in the secondary antibody solution for ~1 h.
  6. Make sure to have TBS solution available before you start. Aspirate the secondary antibody solution off the coverslip and immediately add 150 μL of TBS. Do not let the coverslips dry out during this process.
  7. To complete the post secondary wash, add 150 μL of TBS per coverslip, let incubate at room temperature for 3 min covered, then aspirate.
  8. To add DAPI, dilute the DAPI stain 1:1000 in TBS and add 150 μL DAPI per coverslip.
  9. Incubate at room temperature for 10 min covered, then aspirate.
  10. Add TBS as in Step #2 for the final wash and leave for 3 min. Do not aspirate.
  11. Obtain glass slides from the front laboratory bench and label your slides with all of your experimental information and group name, add one drop (15 uL) of mounting media to the slide.
  12. Aspirate the final TBS wash and using tweezers place the coverslip cell-side down on the mounting media "spot" on the microscope slide. Try your best to avoid bubbles by slowly placing the coverslip over the mounting media.
    • The cell-side of the coverslip is the side that was facing up in the staining chamber.
  13. Complete Steps #5-6 for coverslips from all of the coverslips you stained.
  14. Add one small drop of nail polish to each side of your coverslip to seal it to the glass slide.

In your laboratory notebook, complete the following:

  • Why is it important to wash the secondary antibody from the coverslip before imaging?
  • What stain is used following secondary antibody? What cellular component is stained in this step? And why is this useful?

Part 2: Set up experimental conditions for cytotoxicity assay

Reagents list

  • 4% paraformaldehyde (Electron Microscopy Sciences)
  • Sorbitol-citrate buffer: 1.2M sorbitol (Sigma), 10mM citric acid (Sigma), pH 7.0
  • Zymolase (Zymo Research)
  • permeabilization buffer: 0.1% Tween-20 in Phosphate buffer saline (PBS) (from Invitrogen)
  • blocking buffer: 1% bovine serum albumin (BSA) in PBS (BSA from Sigma)
  • 1:500 Alexafluor-488 conjugated primary antibody to V5 tag, mouse (from Genescript)
  • 1:1000 DAPI (from ThermoFisher)
  • Glycerol (Sigma)
    • Synthetic dropout-uracil (SD-U) media: 0.17% yeast nitrogen base without amino acid and ammonium sulfate (BD Bacto), 0.5% ammonium sulfate (Sigma), 0.192 % amino acid mix lacking uracil (Sigma), 2% glucose (BD Bacto), 0.1% adenine hemisulfate (Sigma)
    • Cadmium chloride (Sigma), [stock] = 100mM

Navigation links

Next day: Confirm transporter expression and cell survival of yeast exposed to metal

Previous day: Analyze ICP-OES data