RRP6 purification for EM
Date: 09/08/20
Related experiment August 19, 2020 - RRP6 purification for EM
Anti-FLAG IP
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG)
Extraction Buffer: 20mM HEPES, pH 7.4, 500mM NaCl, 1% Triton X-100 (v/v)
→ note: In a subsequent round, we could choose to add RNase A/T1 or benzonase in the initial extraction or in subsequent washes to possibly improve homogeneity.
Scale: 250mg with 25ul of anti-FLAG beads
Weigh out 250mg RRP6-3x FLAG; add 1250ul extraction buffer with protease inhibitors to each (1:5, w/v) to each tube
Vortex to mix; put the tubes on ice after resuspending the powder
Sonicate @ 2 Amp, 5x 2 sec; Repeat once (30J total per 250mg sample)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Take the clarified lysate
Set up one 250mg IP reaction
Add 25ul pre-washed beads to 250mg IP reaction (10ul beads per 100mg powder)
Incubate @ 4°C for 1h with rotation (cold room)
Wash beads with 3x 1ml extraction buffer; do all wash steps in the cold room
Transfer the beads to fresh tubes during 2nd wash
The 3rd wash should contain only 0.01% Triton X-100
After the 3rd wash, spin down briefly and remove any remaining liquid
Add 20ul 1mg/ml 3xFLAG peptide in extraction buffer w/ 0.01% Triton X-100 to the beads (1mg/ml 3x FLAG peptide was diluted from 5mg/ml stock in extraction buffer)
Elute for 15 min at RT w/ mixing
Place on a magnet; transfer the supernatant to a fresh tube
Add 10ul extraction buffer w/ 0.01% Triton X-100 to wash the beads
Pool these two fractions together (30ul total per 250mg IP)
Save 2ul 3xFLAG elution for gel analysis (28ul will pass through Zeba desalting column)
Clean up elution using Zeba column and Spin X column
I. Removing 3x FLAG peptide using Zeba 40K column
Remove the storage buffer from Zeba 40K column, spin @ 1000g for 1’
Equilibrate three 75ul Zeba 40K column (sample range: 3-12ul) with 3x 50ul of 20mM HEPES, pH 7.4, 300mM NaCl, NO DETERGENT, spin @ 1000g for 1’
Sample recovery:
Load 10ul of sample on column and spin @ 1000g for 1’ (manufacturer recommendation: spin 1000g for 2’); recover 12ul
Combine sample from 3 columns (32ul total); save 2ul for gel
Spin @ 1000g for another 1’, check the volume recovered (2ul extra)
Combine sample from 3 columns (6ul total); save 2ul for gel (Zeba 40_2nd spin)
Combine the desalted sample (2x14ul=28ul)
II. Sample clean up using Spin X column
Pre-wet Spin X column with extraction buffer to avoid volume loss of the sample
Split the desalted 3x FLAG elution into 2x 14ul aliquots
Save one aliquot aside (without passing through Spin X column)
Pass another aliquot through a 0.22um Spin X column
Save 2ul from each sample (+/- Spin X column) for gel analysis (Sypro stain)
Now the sample should be ready for EM
Add LDS and 50mM DTT (final concentrations) to gel samples
Heat @ 70°C for 10’
Run samples on 15-well 4-12% Bis-Tris gel for Sypro stain
Stop the gel earlier to make sure that 3xFLAG peptide will be on the gel (200v for 30’)
1) Marker_1ul prestained
2) Space
3) 3x FLAG elution_500mM NaCl (2ul, 6% of 250mg IP)
4) Desalted RRP6_before Spin X (2ul)
5) Desalted RRP6 after Spin X (2ul)
6) Space
7) BSA_50ng
8) BSA_100ng
9) Space
10) Marker_5ul prestained
