RRP6 purification for EM
Date: 08/19/20
From John: My suggestion is that we increase the salt to 500mM and we SKIP the gradient altogether - the yield will be markedly increased and the 0.5g will probably have more than enough. We will 3xFLAG elute in 500mM NaCl - then we can clean-up over Zeba column at 250mM (this may not be needed - but should reduce the 3xFLAG peptide present - although it is not clear if that is interfering) and then we can pass through a 0.2um spin-x column - then spot it.
Fig. 1B from Domanski et al 2016
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: we used 300mM NaCl in Domanski, M., Upla, P., Rice, W., Molloy, K., Ketaren, N., Stokes, D., Jensen, T., Rout, M., LaCava, J. (2016). Purification and analysis of endogenous human RNA exosome complexes. RNA 22(9), 1467 - 1475. https://dx.doi.org/10.1261/rna.057760.116
→ we are taking the assumption that the gradient will be dispensable with the higher salt washing
→ 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 each 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 each tube (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 per tube 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
Zeba column
→ note: Zeba 7K should remove 95% of salts and other small molecules (< 1000 MW) and good recovery of proteins and other macromolecules (> 7000 MW). Zeba 40K should recover proteins larger than 40kD and remove molecules smaller than 2000 MW. The MW of 3xFLAG is 2864Da.
→ note: “Our previous results indicate that Zeba does not effectively deplete 3xFLAG peptide. Inconsistencies in P6 measurements using UV cast doubt on effective depletion of P6. P30 effectively depletes FLAG. The only FLAG peptide that may be present would be as fragmented peptide that may only result in increased background in the low mass region of the spectrum.”
This tells me that the zeba 40k might work, but the 7k won’t…We will try Zeba 40K this time
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, 250mM 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)
Spin X column (will do a side by side comparison here to see if spin X column helps)
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
LDS wash of the beads
Wash the beads from 250mg IP with 30ul of LDS
Load 2ul on gel to see what is left on beads after native elution
Add 1x 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) 3x FLAG elution_500mM NaCl (2ul, 6% of 250mg IP)
3) LDS wash of the beads (2ul, 6% of 250mg IP)
4) Desalted RRP6_before Spin X (2ul)
5) Desalted RRP6_Zeba 40K_2nd spin (2ul)
6) Desalted RRP6 after Spin X (2ul)
7) BSA_10ng
8) BSA_50ng
9) BSA_100ng
10) Marker_5ul prestained
