MATERIALS A N D METHODS
CHAPTER 2 MATERIALS AND METHODS NTF
M A A V I L E S I F L K R S Q Q K K K T S P L N F K K R L F L L T V H K L S Y Y E Y D F E R G R R G S K K G S I D V E K I T C V E T W P E K N P P P E R Q I P R R G E E S S E M E Q I S I I E R F P Y P F Q W Y D E G P L Y V F S P T E E L R K R W I H Q L K N V I R Y N S D L V Q K Y H P C F W I D G Q Y L C C S Q T A K N A M G C Q I L E N R N G S L K P G S S H R K T K K P L P P T P E E D Q I L K K P L P P E P A A A P V S T S E L K P W A L Y D Y M P M N A N D L Q L R K G D E SH3R SH2F___ Y F I L E E S N L P W W R A R D K N G Q E G Y I P S N Y V T E A E D S I E MY E W Y S K H M T ^ S Q A E Q L L K Q E G K 276 E G G F I \ ^ D S S K A G K Y T V S V F A K S T G D P Q G V I R H Y W C S T P Q S Q Y Y L A E K H L F S T I P E L I N JP SH2R 361 Y H Q H N S A G L I S R L K Y P V S Q Q N K N A P S T A G L G Y G S W E I D P K D L T F L K E L G T G Q F G W K Y G K 421 W R G Q Y D V A I K M I K E G S M S E D E F I E E A K V M M N L S H E K L V Q L Y G V C T K Q R P I F I I T E Y M A N G 481 C L L N Y L R E M R H R F Q T Q Q L L E M C K D V C E A M E Y L E S K Q F L H R D L A A R N C L V N D Q G W K V S D F 541 G L S R Y V L D D E Y T S S V G S K F P V R W S P P E V L M Y S K F S S K S D I W A F G V L M W E I Y S L G K M P Y E R 601 F T N S E T A E H I A Q G L R L Y R P H L A S E K V Y T I M Y S C W H E K A D E R P T F K I L L S N I L D V M D E E S
Fig. 2.1 Amino acid sequence of Btk. The protein dom ains am plified by the primer pairs (Table 2.2) are indicated by the shaded boxes. The residues m utated in the JP and 276 SH2 dom ains are highlighted.
CHAPTER 2________________________________________________ MATERIALS AND METHODS
2.3
METHODS 2 - ANALYSIS OF FUSION PROTEIN
LIGANDS
2.3.1 Cell culture, stim ulation and lysis
Culture: All cells were grow n in RPMI m edia (Gibco or Sigma) containing 10% PCS (v / v), 5 mM L-glutamine and 10 |L ig / ml ciprofloxacin (Bayer) at 37°C in
5% CO2 to a density of 0.5-1x10^ cells/m l. Cells were pelleted by centrifugation
at 200 g for 5 min, w ashed in pre-w arm ed RPMI (containing no additives) before centrifugation for a further 5 min.
Stim ulation: Cells were stim ulated through the BCR by cross-linking surface p
chains by the addition of 100 p g / ml goat P(ab)2 anti-hum an p fragments. Cells
were incubated at 37°C for the indicated times before lysis.
Non-specific cell stim ulation was perform ed as above except 5 mM M gCh was added in place of the antibody fragm ents (Nakashima et al., 1994).
Lysis: Unless otherwise stated cells were lysed for 10 m in w ith ice-cold NP40 lysis buffer at a concentration of 2x10? cells/ ml. Lysates were centrifuged at 4°C at 15 000 g for 15 m in to rem ove insoluble material.
2.3.2 Fusion protein precipitation
All steps were perform ed at 4°C unless otherwise stated. Lysates from 10^ cells were incubated w ith 10 pg fusion protein attatched to GS-4B beads for 2 h -
0/ n w ith rolling. Beads were precipitated by centrifugation at 1 500 g for 30 s
and w ashed 3x w ith lysis buffer. Precipitates w ere boiled w ith 2x SDS-sample buffer for 5 m in before loading on SDS-PAGE gels.
CHAPTER 2________________________________________________ MATERIALS AND METHODS
2.3.3 Im m unoprécipitation
All steps were perform ed at 4°C.
U sing rab b it polyclonal antiserum : Lysates from 10^ cells w ere incubated w ith approxim ately 1 pg antiserum for 1 h w ith rolling. 10 pi of Gam mabind beads (Pharmacia Biotech) were added and incubated for a further 1 h. Beads w ere w ashed and prepared as described for fusion protein precipitates.
U sing 4G10: Lysates from 10^ cells were incubated w ith lOpl 4G10 mAh covalently attatched to Sepharose beads for 2 h. Beads were w ashed as above. All protein m olecular weights are expressed in kDa. Molecular w eight
m arkers used w ere High m olecular w eight standard (Sigma), Rainbow m arker (Amersham Life Science), Kaleidoscope prestained standards (Biorad).
2.3.4 SDS-PAGE analysis
Resolving gels contained 6-15% (w /v ) acrylamide (Protogel, National
Diagnostics) in 375 mM Tris-HCl, pH 8.8, 0.1% (w / v) SDS. Stacking gels were poured on top of resolving gels and contained 4% acrylam ide in 125 mM Tris- HCl, pH 6.8. Gels were polym erised using 0.1% (w / v) am m onium
persulphate and 0.1% (v /v ) temed.
M inigels (8x5 cm): Precipitates from the equivalent of 3x10^ cells w ere loaded per lane. Whole cell lysates from the equivalent of 7.5x10^ cells were loaded per lane. Samples were electrophoresed across 70-150 V.
Large gels (12x17 cm): Precipitates from 10^ cells w ere loaded per lane. Whole cell lysates from the equivalent of 2.5x10^ cells were loaded per lane. Samples w ere electrophoresed across 40-50 V.
2.3.5 Coom assie b rillian t blue staining
250 m g /L CBB w as dissolved in an aqueous solution containing 40% methanol (v/v), 10% acetic acid (v /v ) solution. Gels were stained for 30 m in before
CHAPTER 2________________________________________________ MATERIALS AND METHODS
destaining w ith an aqueous solution containing 10% m ethanol (v / v), 5% acetic acid (v/v). Gels were vacuum dried.
2.3.6 Protein transfer to nitrocellulose filters
Gels were incubated w ith transfer buffer for 5 m in (minigels) or 20 m in (large gels) and transferred to nitrocellulose filters (BDH) using a Biorad semi-dry blotter according to m anufacturer's instructions. As a general rule minigels w ere transferred for 25 m in at 12 V and large gels for 50 m in at 18 V.
2.3.7 Immunoblotting
Between 0.1 and 1 \ig m ouse monoclonal antibody (mAb) or rabbit polyclonal
antiserum per ml of blotting buffer w ere incubated w ith nitrocellulose filters for 1-3 h at RT. Filters were w ashed 3 times w ith PBSA-T before incubation for 1-2 h w ith horse radish peroxidase (HRP) conjugated secondary antibody. A 1/1000 dilution of rabbit anti-mouse IgG-HRP was used for detection of prim ary mouse mAb and a 1/1000 dilution of goat anti-rabbit IgG-HRP was used for detection of prim ary rabbit polyclonal antiserum. Nitrocellulose filters w ere then w ashed 5 times w ith PBSA-T before incubation for 1 m in w ith chemiluminescence reagent, ECL (Amersham Life Science). Reactive species were visualised by exposure to X-ray film for betw een 5 s and 15 min.
2.3.8 Metabolic labelling
Cells w ere w ashed in RPMI lacking cysteine and m ethionine and resuspended *■
in this m edium containing 20 p C i/m l at 10^ cells/m l. Cells w ere incubated at
37°C in 5% CO2 for 5 h before being w ashed in RPMI and lysed as described in
2.3.1. Precipitated proteins were separated by SDS-PAGE and visualised by autoradiography of dried gels.
CHAPTER 2 MATERIALS AND METHODS
2.2.9 In vitro kinase assays
Fusion protein precipitates and im m unoprecipitates w ere perform ed as described up to the stage w here beads had been w ashed 3 times. Beads were then w ashed in PBSA followed by kinase buffer. Beads w ere then resuspended in 20 pi of kinase buffer containing 5pCi P^p] y A T P/ sample. Reaction mixtures w ere incubated for 20 m in at RT before the addition of 2x SDS loading buffer and boiling for 5 min. Samples were resolved by SDS-PAGE and incorporated phosphate visualised by autoradiography of dried gels or nitrocellulose filters.
2.2.10 Proline peptide competition
Peptide (Research Genetics), see Table 2.3, was incubated w ith 2 pg fusion protein attatched to GS-4B beads for 1 h at 4°C in 100 pi NP40 lysis buffer. 400 pi of Daudi cell lysate containing 1.5x10^ cell equivalents was added such that the final peptide concentration became either 25,125 or 250 pM. Precipitations w ere then carried out as described in section 2.3.2. Samples were resolved by SDS-PAGE and im m unoblotted w ith SK3 anti-WASP antiserum to quantitate bound WASP and anti-GST antiserum to ensure even loading of fusion protein.
Table 2.3 Proline rich peptides derived from the WASP sequence
PEPTIDE SEQUENCE WASP RESIDUES
WP2 RRGGLPPLHPGGDQ 170-185 WP4 RRQEPLPPPPPPSRGG 308-323 WP7 LGIAPPPPTPRGPPPPGR 347-364 WPIO SGNGPAPPPLPPALVP 406-421 Poly-L-Proline (P) n (1 0 -1 0 0 ) 314-319 367-373 Control IFFYQSPYDSEQVPA - 79