RRP6 purification with 300mM buffer followed by glycerol gradient
Date: 03/09/21
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG) from Leila
Extraction Buffers: 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v)
Anti-FLAG beads used in this experiment were made with anti-FLAG antibody from Marty (beads made on 09/03/20)
Scale: 8x 250mg IP with 25ul of anti-FLAG beads per 250mg IP
Weigh out 8x250mg RRP6-3x FLAG; add 1250ul extraction buffer 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, 4x 2 sec, (2 sec pulse on, 1 sec pulse off; energy: 15J); Repeat once (30J total per 250mg sample)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Take the clarified lysate
Set up 8x 250mg IP reactions (25ul beads per 250mg powder)
IP @ 4°C for 1h with rotation (cold room)
Prepare 1mg/ml 3xFLAG peptide for native elution (20ul per 250mg reaction) Dilute 5mg/ml stock with 300mM extraction buffer
After 1hr IP, wash beads with 2x 1ml extraction buffer; do all wash steps in the cold room
Transfer the beads to fresh tubes during 2nd wash
After the 3rd wash, spin down briefly and remove any remaining liquid
Add 20ul 1mg/ml 3xFLAG peptide in extraction buffer
Elute for 15 min at RT w/ mixing
Place on a magnet; transfer the supernatant to a fresh tube
Add 10ul of extraction buffer to each tube to wash the beads
Collect the wash and pool elution and wash from all 8 tubes together (8x20+8x10=240ul total)
Keep the samples on ice before loading the gradient
Prepare 10% and 40% glycerol
Gradient solution – ‘light’
10% v/v glycerol
20 mM HEPES-NaOH, pH 7.4
100 mM NaCl
Sterile filter with a 0.45 μm syringe filter
Gradient solution – ‘heavy’
40% glycerol
20 mM HEPES-NaOH, pH 7.4
100 mM NaCl
Sterile filter with a 0.45 μm syringe filter
03/09/21
Run one 26-well 4-12% Bis-Tris gel, one for silver stain
Take 15ul from each of the fractions listed below, 0.5ul from the input
Add LDS and 50mM DTT
75°C for 10’
After heating, spin @ top speed
Run 4-12% Bis-Tris gel followed by silver stain
1) Marker_0.5ul prestained
2) Space
3) Input RRP6_300mM NaCl (0.5ul)
4) Space
5) Fraction2_RRP6 (15ul)
6) Fraction4_RRP6
7) Fraction6_RRP6
8) Fraction7_RRP6
9) Fraction8_RRP6
10) Fraction 9_RRP6
11) Fraction10_RRP6
12) Fraction11_RRP6
13) Fraction12_RRP6
14) Fraction13_RRP6
15) Fraction14_RRP6
16) Fraction15_RRP6
17) Fraction16_RRP6
18) Fraction17_RRP6
19) Fraction18_RRP6
20) Fraction19_RRP6
21) Fraction20_RRP6
22) Space
23) BSA_5ng
24) BSA_25ng
25) Space
26) Marker_2.5ul prestained (not shown)
Passing the glycerol fractions through Zeba 7K to get rid of glycerol
Take fractions 9 and 10 (~200ul each); save 10ul for negative stain; save 15ul from each before desalting (glycerol fraction) for gel analysis
Remove the storage buffer from Zeba 7K column, spin @ 1500g for 1’
Equilibrate six 0.5ml Zeba 7K column (sample range: 30-130ul) with 3x 300ul of 20mM HEPES, pH 7.4, 250mM NaCl, NO DETERGENT, spin @ 1000g for 1’
Load 85ul of sample on column and spin @ 1500g for 2’; recover 85ul; combine samples from same fraction
Save 15ul for gel analysis; Keep 70ul for EM (single desalted samples)
Load 85ul of single desalted sample on another equilabrated 0.5ml Zeba 7K column and spin @ 1500g for 2’; recover 85ul
Save 15ul for gel analysis; Keep 70ul for EM (double desalted samples)
Run sample collected from each step side by side on 4-12% Bis-Tris gel followed by silver stain
1) Marker_0.u2l prestained
2) Space
3) Input RRP6_300mM NaCl (0.5 ul)
4) Space
5) Fraction 9_glycerol
6) Fraction 9_desalt1x
7) Fraction 9_desalt2x
8) Fraction 10_glycerol
9) Fraction 10_desalt1x
10) Fraction10_desalt2x
11) Space
12) BSA_5ng
13) BSA_25ng
14) Space
15) Marker_2ul prestained (not shown)
