RecA-filament affinity chromotography of LexA
Notes:
Use entire 75 mg vial of oligo-dT-cellulose from FastTrack 2.0 mRNA isolation kit.
RecA MW = ~40 kD
For wash steps, add solution, spin beads 3000 g/5’ and pipet off supernatant.
Use cut 1 ml tips to transfer beads.
Column volume of RecA-filament beads ~250 l
1. Wash oligo-dT-cellulose beads with 1 ml 0.25X RecA rxtn buffer
2. Prep of RecA-filament beads (follow order for addition to washed beads):
917 l
H2O
25 l
10X NEB RecA reaction buffer
20 l
10 mM ATP[S]
(0.2 mM final)
37.8 l
RecA NEB
(75 g; 2 mM final)
o
Inc 4 C O/N inverting
3. Place ~30 ml acetone at -20 oC in glass container sealed in plastic bag.
4. Centrifuge beads and save supe as RecA “flow through”.
5. Wash beads with 1 ml 0.25X RecA buffer.
6. Resuspend w/ 1.5 ml 0.25X RecA buffer and split into 3, 1.5 ml tubes; ~680 l/tube.
7. Add 90 g LexA in 500 l LexA storage buffer to each tube containing filaments.
8. Invert at room temperature 1 hr 15 min
9. Pour 3, 10-well gels (15% to resolve full-length from digested LexA; 12% for K156A
mutants)
10. Make 1.6 ml each elution buffer:
62.5 125
250
500
1M
[NaCl] final
40
40
40
40
40
l 10X RecA buffer
20
40
80
160
320
l 5 M NaCl
1540 1520 1480 1400 1240 l H2O
(or 2x: 770
760
740
700
620
l H2O)
11. Transfer beads to 5 ml column and add 1.25 ml 0.25X RecA buffer
12. Collect “wash” in 15 ml tube
13. Apply 500 l of each elution buffer and collect elution in 1.5 ml tube
14. Acetone ppt protein in 2 ml tube for Silver Stain gel:
400 l elution protein sample
1600 l -20 oC Acetone
Vortex and inc -20 oC 1hr
Cent 10’/ 13K-15K g
Aspirate and dry pellet r.t. 30’
Resuspend in 20 l 1X sample buffer; boil 5-10’ or 65 oC 10’
For RecA flow through sample: 54 l + 18 l 4X sample buffer
For Purified LexA sample: 900 ng + 20 l 1X sample buffer
For beads sample: Add 150 l 0.25X RecA buffer; remove 18 l + 6 l 4X
For MW marker: load 0.375 l and then 10 l 1X sample buffer
Quickly load gel as follows (omit purified LexA for 9-well gels):
pur. FT
0
62.5 125
250
500
1M
MW LexA RecA Wash 1
2
3
4
5
beads
0.375λ 20 λ 20 λ 20 λ 20 λ 20 λ 20 λ 20 λ 20 λ 20 λ
+10 λ
10X RecA reaction buffer: 70 mM Tris-HCl pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol
LexA storage buffer: 10 mM PIPES-NaOH pH 7.0, 0.1 mM EDTA, 200 mM NaCl, 10%
glycerol
Notes from literature:
Dispensing the Resin
When pipetting streptavidin-agarose, we recommend using large bore, disposable plastic pipette tips. To
ensure reproducible dispensing, cut 3 mm from the end of each pipette tip before use.
equilibrate the resin with the buffer that will be used in the experiment immediately prior to use.
Smith, Little PNAS91 protein sequencing:
1.
Used tac promoter
2.
Suspend cell pellet in 1% Triton X-100/5 mM EDTA/1M KCL/0.016%
lysozyme, inc 4oC 30 min & freeze thawing
3.
Cent 37,000 g 30 min
4.
Mix supe with -LexA; 140 ul supe w/ 25 ul
5.
Ppt with protein A-Sepharose
6.
Elute w/ SDS/PAGE sample buffer and resolve on 15% gel
7.
Trasfer to Immobilon-P membrane
8.
Stain with Coomassie blue and submit C fragment to Edman degradation
Moreau, Devoret MGG82 Slab gel electrophoresis to measure RecA protein
synthesis:
1.
Labeled cells pelleted and resuspend in 25 ul 10mM Tris-HCL pH 7.9/5 mM
MgCl2/0.1 mM EDTA/0.1 mM dTT/50 mM KCl/0.6 mg lysozyme per ml
2.
Freeze thaw 6X
3.
Add 140 ul 50mM Tris-HCL pH7.5/2 M KCl/2% Triton X-100/0.66%
polymin P and inc 0oC 30 min w/ gentle agitation
4.
Pellet debris and DNA in minicent for 15 min
5.
140 ul supe mixed in small glass tubes w/ 25 ul Tris-HCL pH7.5/2 M KCl/2%
Triton X-100/5% anti-RecA
6.
Inc 1h w/ vig agitation and then add 30 ul protein-A-Sepharose CL4B(Pharmacia) and cont inc w/ agitation for >1h
7.
Pour suspensions into small columns and wash with 2ml cold 0.1M Tris-HCL
pH 6.8/0.5M LiCl/1%b-ME
8.
Elute w/ 40 ul 125 mM Tris-HCL pH 6.8/2%SDS/10% glycerol/10%bME/0.0015% bromophenol blue at 45oC, heat 100oC 1 min and run SDSPAGE gel at 30 mA for 4h
9.
10.
Fix proteins 1h w/ 50% Methanol-10%acetic acid
Dry gels and exopose to film for ~4 d.
Shinagawa, Nakata PNAS88 IP:
1. Exponential cells in M9 @ OD600 0.2 labeled with [35S]Meth and MMC 1 ug/ml
were added
2. Prepare cell lysate and ppt w/ antiserum and protein A-Sepharose (Sigma)
3. Wash complexes with RIPA buffer (10% Triton X-100/1% Sodium
Deoxycholate/0.1% NaDodSO4/0.15M NaCl/0.05 M Tris-HCL pH 7.2)
4. Run NadDodSO4/PAGE
Freitag and McEntee (JBC88)
DNA was present at a 20-fold molar excess (nucleotides) to RecA protein.