we did a 24-codition IP screening 21/10/2021 24 IP screening, the results showed tween 20 and brij-58 is not good for e-cadherin extraction. so in this experiment, we select two new detergents, DDM and C12E8, to test the affiniy ability. and also compared Ca+ vs Ca-, NaCl vs KAcet
Material:
link to material summary: E-Cadherin Materials
Cell powder:
50mg BG1 CDH1-HA
50mg*9 CDH1-HA WT
Bead:
Pierce HA bead
Buffer:
| # | Buffer component | pH | detergent | Additive |
| 2 | 20mM HEPES | 7.4 | 1% TRIT X100 | 250mM NaCl |
| 3 | 20mM HEPES | 7.4 | 1% TRIT X100 | 250mM NaCl,4mM Ca |
| 6 | 20mM HEPES | 7.4 | 1% TRIT X100 | 250mM KAcet |
| 7 | 20mM HEPES | 7.4 | "0.5% TRIT X100 0.1% Tween20" | 250mM NaCl, 2mM MgCl2, 20mM KAc, 4mM Ca |
| 33 | 20mM HEPES | 7.4 | 1% ddm | 150mM NaCl |
| 34 | 20mM HEPES | 7.4 | 1% ddm | 250mM NaCl |
| 35 | 20mM HEPES | 7.4 | 1% c12E8 | 150mM NaCl |
| 36 | 20mM HEPES | 7.4 | 1% c12E8 | 250mM NaCl |
| 37 | 20mM HEPES | 7.4 | 1% TRIT X100 | 250mM NaCl, 2mM MgCl2, 20mM KAc, 4mM Ca |
IP
50mg powders with 25ul bead
Protocol: June/2021 IP for BG1 with/without TMTC KO
17. take out 4 ul of elution for silver stain. another 16ul of elution for LC/MSMS
LC-MSMS
Sample preparation: MS sample preparation using S-Trap Micro Ultra-High Recovery protocol rev. 05/27/21
Resuspension solvent: 94.9:5.0:0.1 water:MeOH:FA
Resuspension volume: 25 uL
Injection volume: 5 uL
Link to Raw data:
Link to MaxQuant output:
Silver stain
