LaCava Research Wiki

Beads were conjugated with the following antibodies:
Antibodies:
Marty Taylor anti-FLAG M2, Sabatini Lab Concentration: ~17mg/ml Sigma F3165: Anti-FLAG Clone M2 mouse monoclonal antibody Concentration: ~3.5mg/ml

Cell lines:
SMAD-3xFlag (~65 kD) STAT3-3xFlag (new) (~88kD) 100mg scale with 10ul beads/reaction

Controls:
-Old Marty / Sigma Eriba beads -Hua NY beads

Extraction/Wash Buffer:
20mM HEPES 300 mM NaCl 0.5% Triton

Test DMP XL in both sets of new beads Test RT (25C, 10 min) LDS elution

For each cell line (SMAD4 and STAT3), produce the following samples:

1. Old beads Marty
2. Old beads Sigma
3. New beads Marty
4. New beads Sigma
5. Hua beads
6. New beads Sigma RT elution
7. Hua beads RT elution
8. New beads Sigma XL (70C elution)
9. Hua beads XL (70C elution).
   

1-5 all normal 70C LDS elution, and then test the best new beads we have (new sigma) against our gold standard (Hua) for both the RT elution and the XL.

DMP Cross-linking
DMP should be kept in a desiccator at 4C. Always allow the crosslinking agent to equilibrate to RT before opening. DMP is not recommended for use after storage for longer than 1 month after opening. Old DMP may become hydrolyzed by condensed atmospheric moisture and result in incomplete XL. All solutions should be at room temperature.

1. Wash Dynabeads with 1ml of PBST, then remove PBST with pipette; Repeat once
   
2. Resuspend beads in 600ul of 20mM DMP in Borate buffer (2ml per reaction with 100ul of beads; make this right before use when beads are washing)
3. Perform XL reaction @ RT on the wheel for 30’.
4. After 30’, remove the XL solution
5. Wash beads with 1ml of PBST Goal here is to remove any unreacted free XLinker
6. Wash beads again with 1ml of TBST for 5’ @ RT The goal here is to cause tris to react with any XLinker that reacted on one side with the Ab, but on the other side did not react.
7. Wash beads again with 1ml of PBST
8. Wash beads with 1ml of 1x PBS
9. Wash beads with appropriate lysis buffer prior to IP

Anti-FLAG IP

Weigh out 9x of HEK293_SMAD4_3xFLAG or HEK293_STAT3_3xFLAG

Add 400ul of extraction buffer plus protease inhibitor to each tube (1:4 w/v), vortex to mix

Sonicate 5 x 2 sec at 4 Amp;

Spin @ 20k rcf, 4°C for 10’

Collect the supernatant; save 20ul for potential WB

Set up IP reactions with 10ul of beads each

IP @ 4°C for 30’

After IP, wash 3x 1ml extraction buffer

Switch beads to fresh tubes at 2nd wash step

Elute the beads with 10ul of 1.1x LDS, 70 °C for 5’ with mixing; or RT for 10’ with mixing

Collect the eluate

Add DTT to 50mM to all samples and heat @70 °C for 10’

Run 4-12% Bis-Tris gel to analyze the elution, Blue Silver Stain