Co-immunoprecipitation and western blotting

Crude cell extraction from Drosophila Upd-tumor testes

Materials:

• 1M Tris-HCl (pH8.0)
• 5M NaCl
• 0.5M EDTA (pH8.0)
• 50% Glycerole
• 10% NP-40
• PhoSTOP (Roche, cat No: xxx; store at -20˚C)
• EDTA-free Protease Inhibitor cocktail (Roche, cat No: xxx; store at -20˚C)
• 300 mM PMSF stock in 100% isopropanol (store at RT)

• Lysis buffer [20 mM Tris-HCl (pH8.0), 100 mM NaCl, 2 mM EDTA, 10% Glycerole, 0.2% NP-40,
  1x solution of PhoSTOP cocktail (Roche), 1x solution of cOmplete EDTA-   

  free protease inhibitor cocktail (Roche)and 1 mM PMSF.

  Add reagents shown in red freshly just before use!

• Schneider’s Drosophila Medium (Gibco)
• 1x PBS(-)

• 7cm pestle with microtube (KIMBLE)

   

  Methods:

1. Dissect DrosophilaUpd-induced tumor testes in Schneider’s Drosophila Medium  and put these into 1.5 ml tube with 1 ml of ice-cold 1xPBS(-) (50 testes/tube).
2. Wash the testes with ice-cold 1xPBS(-) three times.
3. Remove 1xPBS(-) completely, freeze the testes by liquid nitrogen and store at -80˚C until we collect enough number of testes [500 µg crude cell extract (derived from 200 pairs Upd-testes)/IP sample).
4. Homogenize testes in 200 µl lysis buffer on ice using plastic pestle.
5. Incubate the homogenized testes on ice for 20 min.
6. Vortex the homogenized testes at highest setting for 15 sec.
7. Centrifuge the sample at 14,000 rpm for 10 min at 4˚C.
8. Take the supernatant (cytoplasmic fraction) and keep the tube on ice.
9. Add 50 µl of lysis buffer containing 100 mM NaCl to the nuclear pellet.
10. Incubate for 60 min on ice with vortex at highest setting for 15 sec in every 10 min intervals to collect nuclear fraction.
11. Centrifuge the sample at 14,000 rpm for 30 min at 4˚C.
12. Take the supernatant (that contains nuclear fraction) and mix the nuclear fraction with the cytoplasmic fraction deribed from the step 8. Keep the tube on ice while protein concentration is measured by BCA assay.
13. Measure protein concentration of the extract by BCA assay

BCA assay 

Materials: 

• Pierce^TM BCA Protein Assay Kit (Thermo Scientific; cat no: 23225)
• 96-well microtest plate (flat bottom)

• Spectrophotometer

   

Methods:

Follow the instruction of Pierce^TM BCA Protein Assay Kit. 

1. Prepare BCA working solution (WR). Required volume of WR is calculated by following formula: (number of standards + number of your smaples) x 2 replicates x 200 µl (volume of WR per sample).
   e.g. In the case you have 2 samples, you need 4 ml of WR from the following formula: (8+2) x 2 x 200 (µl)
   Mix 5 ml of Solution A with 100 µl of Solution B.
2. Make BSA standard series as following:
                                       dH₂O (µl)        BSA (µl)
   1. (2000 µg/ml)           0                    30.0
   2. (1500 µg/ml)        12.5                 37.5
   3. (1000 µg/ml)        32.5                 32.5
   4. (750 µg/ml)           17.5             17.5 of 2
   5. (500 µg/ml)           32.5             32.5 of 3
   6. (250 µg/ml)           32.5             32.5 of 5
   7. (125 µg/ml)           32.5             32.5 of 6
   8. (0 µg/ml)               30.0                    0
3. Make dilution of your sample. The extract from 200 pairs of Upd-induced testes should be diluted as 5 times by 2% SDS/dH₂O (e.g. mix 10 µl extract with 40 µl of 2% SDS/dH₂O).
4. Apply 10 µl of standards and your samples to a 96-well microtest plate.
5. Add 200 µl of WR to each well of standards and your samples.
6. Incubate the 96-well microtest plate at 37˚C for 30 min.
7. Read absorbance at 562 nm by plate reader (locates at David Ginsberg’s lab)
8. Open application on desktop called ‘softmax pro’
9. From ‘protocol’ tab, select ‘protocol manager’->’protocol library’->’Protein Quant’->’BCA’
10. After reading the absorbance, export the data.

Immunoprecipitation

Materials:

• Immunoprecipitation Kit Dynabeads® Protein A (novex; cat no: 10006D)
• Magnetic stand (NEB)

Methods:

• Pre-clearing crude cell extract

1. Resuspend the Dynabeads® Protein A in the stock vial (vortex for >30 sec)
2. Transfer 50 µl Dynabeads® Protein A (for one sample amount, if you have two samples, take 100 µl) to tube. 
3. Place the tube on a magnetic stand to separate the beads from the solution, and remove the liquid phase of the Dynabeads® solution .
4. Wash the beads in 100 µl of ice-cold lysis buffer containing once and place the tube on the magnetic stand to remove the buffer.
5. Add 200 µg crude cell extract (in 200-300 µl lysis buffer) to the tube containing Dynabeads® Protein A.

6. Pipet well and incubate on a rotator at 4˙C for 1 hour.

    

• Incubation with antibody

1. Place the tube on a magnetic stand to separate the extract from the beads (discard the beads).
2. Transfer the extract to a pre-chilled new tube and add 10 µl anti-CG2199 antibody (10 times dilution from the original serum; generated as described above) or 10 µl of pre-immune guinea pig serum (10 times dilution from the original serum), respectively. 

3. Mix well and incubate on a rotator at 4˙C for 1 hour.

    

• Incubation with Dynabeads® Protein A-antibody complex

1. Prepare Dynabeads® protein A
2. Add 50 µl of Dynabeads® Protein A freshly prepared from the step 1-4 to the tube in the step 8.

3. Incubate on a rotator at 4˙C for 1 hour.

    

• Post-IP wash

1. Place the tube on a magnetic stand and remove the supernatant.
2. Wash the beads three times using 200 µl washing buffer (attached in the IP kit).
3. Bring the washed beads (filled in washing buffer) to the MS Bioworks with placing the tubes on ice

References: 

• Nuclear extraction protocol from Thermo Fisher Scientific website (for crude cell extraction): https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-preparation-protocols/nuclear-extraction-method-.html
• Protocol from Immunoprecipitation Kit Dynabeads® Protein A (for IP): https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017347_dynabeads_protein_a_ip_kit_PI.pdf
• Protocol of immunoprecipitation using Protein A/G magnetic beads from NEB website (for IP): https://www.neb.com/protocols/1/01/01/immunoprecipitation-using-protein-ag-magnetic-beads

Western Blot Protocol

Part 1: Protein Sample Preparation

Solutions preparation:

• 1x PBS (in 4C room).
• 1x Proteinase Inhibitor (P.I.) solution: 
  • Put one Proteinase inhibitor cocktail tablet (cOmplete, EDTA-free, 11873580001, at 4C fridge https://www.sigmaaldrich.com/catalog/product/roche/coedtafro?lang=en&region=US) into 1 ml 1xPBS to make 50x P.I. solution. Store 50x P.I. at -20C.
  • Dilute 50 times into 1xPBS to make 1x P.I. right before use (150 ul 1x P.I. for each sample for 10-15 larvae).
• 2x sample buffer:

  • Add âME (2-mercaptoethanol, in safety hood) to 2x Laemmli Sample Buffer (https://www.bio-rad.com/en-us/sku/1610737-2x-laemmli-sample-buffer?ID=1610737) at 1:20 ratio (50 ul to 950 ul for 1 ml). Dilute sample 1:1 with sample buffer.

     

1. Prepare adult/larval tissues.
   • Larva:
     • For each genotype/condition, pick 10-15 larvae into a 1.5 mL EP tube.
     • Rinse larvae twice with 1ml 1xPBS.
   • Testis:
     • For each genotype/condition, dissect 25 0-5d males into 1x PBS (1mL in a 1.5 mL EP tube, special size of EP tube for homogenization) on ice. Don’t leave testes in PBS for more than 45 min.
2. Remove all PBS.
3. Snap freeze tissues in liquid N2 (for around 10s). After this step, you can store your samples at -80C immediately for a later use or directly proceed into next steps.
4. For each sample, immediately add 150 ul 1x P.I. Then smash tissues thoroughly.
5. After smashing all samples, for each sample, add 150 ul 2x sample buffer (containing® ME).
6. Heat at 95C for 3-5min.
7. Put samples on ice for several minutes.
8. Store at -80C.

Part 2: Gel Electrophoresis

Solutions preparation:

• 10x Running/Transfer buffer (for 1 L):
  • 30.3 g Tris Base, Molecular Biology Grade (https://www.sigmaaldrich.com/catalog/product/mm/648310m?lang=en&region=US).
  • 144.0 g Glycine.
• 1x Running buffer (for 1 L):
  • 100 ml 10x Running/Transfer buffer
  • 10 ml 10% SDS
  • 890 ml milliQ water
• 1x Transfer buffer (for 1 L. We don't use this transfer buffer since we use iBlot 2 Dry Blotting System https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/iblot-dry-blotting-system.html):
  • 100 ml 10x Running/Transfer buffer
  • 200 ml Methanol
  • 700 ml milliQ water
• 10x TBS buffer (for 1 L, stable at 4 °C for 3 months):
  • 24 g of Tris Base
  • 88 g of NaCl
  • Dissolve in 900 mL distilled/sterile/milli Q water
  • Adjust pH to 7.6 with HCl
  • Adjust final volume to 1 L with Milli-Q Water
• 1x TBST buffer (for 1 L, at RT):
  • 100ml 10x TBS
  • 900ml DW
  • 1ml Tween-20 (use stir bar to mix detergent well).

1. Thaw protein samples on ice.
2. Assemble gel electrophoresis tank. Gently remove the comb of the gel, remove the plastic seal at the bottom and assemble it into the tank.
3. Pour 1x running buffer into the inner space of the tank, the level should cover the gel top but do not over the tank wall. Pour 1x running buffer into the outer space of the tank, achieve around 1/3 of the tank height.
4. Load protein ladder and samples: for 12% 10-well gel, each well has a maximum volume of 30 ul. Load 5 ul ladder (BIO-RAD #161-0374) and 15 ul for each protein sample (avoid using edge wells for samples because they are easily bended).
5. Run at 125V for 1-2 hours (check every 10 min after 1 hour, it's ready when the bromphenol blue band is near the gel bottom). The current would be 0.02A when there is only 1 gel, 0.04 A when there are 2 gels.

Part 3: Transferring

1. Prepare 100 ml milliQ water and 100 ml 20% EtOH (20 ml 200-proof (100%) or 190-proof (95%) EtOH with 80 ml milliQ water).
2. When gel is ready, soak gel in 20% EtOH for 10 min.
3. Set up blot sandwich (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/iblot-dry-blotting-system.html)
4. Select template, usually choose P0, and start. This takes 7 min.
5. Take out the membrane, cut the membrane with the region transferred.
6. Rinse the membrane in 1x TBST twice.

Part 4: Blotting

1. Prepare 5% Blocking solution: 1 g Nonfat dry milk (BioRAD Blotting-Grade Blocker) in 20 ml TBST, stir until complete resolving.
2. Block at RT nutator for 1 hour, speed at 65.
3. Rinse with TBST for 5 min. (keep timing strict)
4. Cut the membrane at appropriate site for different antibody staining.
5. Incubate with rotation the membrane with appropriate dilutions of primary antibody in 1x TBST overnight at 4°C. Use 10 mL TBST in big box, 6 mL TBST in small box. (1:10,000 dilution of serum for primary Covance antibodies; 1:3,000 dilution for commercial antibodies such as tubulin, etc. Adjustment might be considered.)
6. Rinse the membrane with 1x TBST once. Wash the membrane in 1x TBST for 3 times, 5 min each. Use 10 mL TBST for big box, 6 mL TBST for small box. Keep the washing time strict.
7. Remove TBST, then add secondary antibody. (1:10,000 dilution. Big box: 10 mL TBST + 1 uL Ab; small box: 8 mL + 0.8 uL Ab) (HRP-conjugated secondary ABs are stored together in -20 C with fluorophore tagged 2^nd Abs)
8. Incubate with rotation for 1 hour at RT. 
9. Rinse the membrane with TBST.
10. Wash the membrane in 1x TBST for 3 times, 10 min for each.
11. Prepare substrate: 1.5 mL + 1.5 mL for each membrane piece. Warm up substrate at RT before using.
12. After washing, put membrane piece on the plastic film. Add 2.5 mL substrate, incubate for 1 min at RT.
13. Acquire images using darkroom development techniques. The amount of loaded sample in gel electrophoresis and primary antibody concentrations are two main factors that will affect the final signal.