IP protein for mass spec analysis of PTM

Treat cells with
10nM Calyculin A
100uM Sodium orthovanadate
2.5mM Nicotinamide
2.5mM Sodium butyrate
30min 37C

Harvest cells from 5 15cm dishes
Resuspend in cold PBS and transfer to eppies
Spin 1800rpm 5min
Wash again with cold PBS. 4 plates of cells/ tube
Remove PBS and lyse with 500ul protein lysis buffer + 1% protease inhibitor cocktail + phosphatase inhibitors
Lyse on ice with occasional vortex 2h

Prepare Dynabeads (Invitrogen)
Completely resuspend Dynabeads by rotating for 5min or vortex 5s
Transfer 50ul beads to eppie
Separate on magnet until supernatant is clear and remove supernatant
Remove tube from magnet

Bind Antibody
Add antibodies (20-30ul of M2 for each IP) up to 200ul Ab binding and washing buffer
Incubate with beads 1h 4C rotate
Place tube on magnet and remove supernatant
Remove tube from magnet and resuspend beads in 200ul Ab Binding and Washing buffer. Wash by gentle pipetting

Immunoprecipitation
Place tube on magnet and remove supernatant
Add lysate to beads and gently pipette to resuspend the beads (keep 5ul as input to run gel)
Incubate O/N 4C rotate

Wash
Place tube on magnet. Transfer supernatant to clean tube (keep for gel)
Wash beads 4x in 1ml TNMEN-150. Mix on shaker 5min 4C
Keep all wash for gel
Resuspend beads-Ab-Ag complex in 100ul Wash buffer and transfer to clean tube to avoid co-elution of proteins bound to tube wall.

Elution
Place tube on magnet and remove supernatant
Add 20ul elution buffer (50mM glycine pH2.8) and 4ul 6x SDS sample loading buffer. Pipette to resuspend
Boil 5min
Store all washes, superatant, IP products at -20C

Run gel
2ul Input, 22ul IP for Coomassie stain
2ul Input, 2ul IP for Western

To stain Coomassie
Rinse peptide gel in ddH2O
Fix gel in 40% Methanol, 10% Acetic aci 30min
Add 50ml Coomassie safe or enough to cover gel. Shake 20min-1h
Wash in ddH2O 2h. Bands will appear during wash
Store in water

To prepare for mass spec
Cut band from Coomassie stained gel with fresh blade
Store in clean tue in 1% acetic acid

Protein lysis buffer
400mM NaCl
10mM HEPES
0.1mM EGTA
0.5mM DTT
5% glycerol
0.5mM PMSF
1% TritonX-100

Phosphatase inhibitors
1% Sigma phosphatase inhibitor 2
1% Sigma phosphatase inhibitor 3
5mM sodium butyrate

TNMEN-150
50mM Tris-HCl pH8
0.5% NP40 (Igepal)
1mM EDTA
2mM MgCl2
150mM NaCl
Add protease inhibitor and phosphatase inhibitors before use

Whole mount in situ hybridization

Fix embryos O/N 4% PFA/DEPC PBS
Wash 2x 5min DEPC PBS
Dehydrate through 50% MeOH/ PBS DEPC, then 2 changes of 100% MeOH
Store -20C
Day 1 (DEPC all solutions): Pretreatment and Hybridization
From dehydrated embryos in MeOH, rehydrate through 75%, 50%, 25% MeOH/ PBST. Just let embryos sink, don't need to rock
Wash PBST 2x 5min
Treat with 5ug/ml Proteinase K in PBST 5-20min depending on size (for chick ~ 1min/ stage)
Remove proteinase, rinse briefly with PBST
Post-fix with 4% PFA + 0.1% Glutaraldehyde/ PBST
Rinse with PBST
Rinse once in 1:1 PBST/ hybridization mix. Let embryos settle
Rinse with 1ml hybridization mix. Let embryos settle
Replace with 1ml hybridization mix and incubate horizontally 1h 65C
Add 1ml pre-warmed hybridization mix with 1ug/ml DIG-labelled RNA probe. Incubate horizontally at 65C. Rotate.
Day 2 (Don't need to DEPC): Post-hybridization washes
Rinse twice with 65C hybridization mix
Wash 4x 30min 1.5ml prewarmed hyb mix 65C
Wash 10min 1.5ml prewarmed 1:1 hyb mix/ MABT 65C
Rinse 2x 1.5ml MABT
Wash 1x 15min 1.5ml MABT RT
Incubate with 1.5ml MABT + 2% Boehringer Blocking Reagent (BBR) RT 1h
Incubate with 1.5ml MABT + 2% BBR + 20% heat-treated sheep serum RT >1h
Incubate with 1ml 1ml MABT + 2% BBR + 20% serum + 1:2000 AP-anti-DIG Ab O/N 4C

Day 3: Washes
Rinse 3 times with 1.5ml MABT
Wash 6-8x 1h with 2ml MABT
Wash O/N 4C

Day 4: Development
Wash 2x 20min with NTMT
Incubate BM Purple AP substrate RT dark
Check after 30min
When ready, rinse with PBST and wash 2x
Postfix in 4% PFA 2h RT
Rinse with PBSt and wash twice
Store 4C

PBST
PBS + 0.1% Tween 20

Hybridization mix50% Formamide
1.3x SSC
5mM EDTA, pH 8
50ug/ml Yeast tRNA
0.2% Tween 20
0.5% CHAPS
100ug/ml Heparin
DEPC H2O

Hybridization mix
Formamide  25ml
20X SSC pH5 with citric acid  3.25ml
0.5M EDTA pH8  0.5ml
20mg/ml Yeast tRNA   125ul
10% Tween 20   1ml
10% CHAPS   2.5ml
50mg/ml Heparin   100ul
H2O   17.5ml
Total  50ml

5x MAB
Maleic acid  11.6g (pH to 7.5 before adding NaCl, neutralize with 10N NaOH, careful after pH 5)
NaCl  8.7g
H2O  185ml
Total  200ml
Dilute with H2O and add 0.1% Tween 20

10% Boehringer Blocking reagent
10% in MAB.
Heat to dissolve. Autoclave. Aliquot and freeze
NTMT (make fresh)
5M NaCl  1ml
2M TrisHCl pH9.5  2.5ml
2M MgCl2   1.25ml
10% Tween-20   5ml
H2O   40.25ml
Total  50ml

Cryo-section In Situ Hybridization

Day 1 (DEPC solutions):
Thaw slides
Post fix in fresh 4% PFA/ DEPC PBS 10min. Save the PFA
Wash 2x 5min DEPC PBS
Incubate 2min in 5ug/ml Proteinase K/ DEPC PBS (Time sensitive)
Wash 5min DEPC PBS
Fix in 4% PFA from before 5min
Wash 5min DEPC PBS
Acetylate sections for 10min with acetic anhydride + 0.1M TEA-HCl pH7.5. Add acetic anhydride to TEA immediately before use.
Wash 5min DEPC PBS
Dehydrate 5min in 70% EtOH, 2min in 95% EtOH
Air dry comepletely
Wash 30-45min in Tris/Glycine Buffer
Prepare hybe mix 150-200ul/ slide
Add 1ug/ml probe
Heat hybe with probe to 80-90C, store on ice
Add 150-200ul hybe with probe onto the slide, cover with a hybrislip
Hybe O/N at 65C. Add damp paper towels to prevent drying

Day 2 (No need to DEPC):
Wash 3x 20min 5x SSC RT
Wash 40min 60C 0.5x SSC 20% formamide
Change wash and place at RT until 37C
Wash in NTE 15min 37C
Wash in NTE + 10ug/ml RNase 30min 37C
Wash in NTE 15min 37C
Wash in 0.5x SSC 20% formamide 30min 60C
Wash 2x SSC 30min RT
Place in 1% blocking solution for 10min at least
Add anti-DIG 1:5000 in blocking solution
Incubate O/N at 4C in humidified chamber

Day 3:
Wash slides 4x 10min, 1x 20min in TBS
Wash slides 10min in 0.1% Tween-20, 0.5mg/ml levamisole hydrochloride in water
Warm BM Purple to RT, mix
Add 10ul of 100x levamisole/ tween-20 per 1ml of BM purple
Place about 170ul of staining solution per slide. Cover with hybri-slip. Incubate RT dark.
Stop reaction by washing slides 10min PBS +1mM EDTA
Dry slides mount with DAKO mounting media.

TEA-HCl pH8
Triethanolamine-HCl  3.71g
DEPC H2O  199ml
10N NaOH  900ul
Total  200ml

Acetic anhydride/ TEA-HCL
Acetic Anhydride  125ul
0.1M TEA-HCl pH7.5  50ml

Tris/Glycine buffer
Tris base  24.2g
Glycine  15g
DEPC H2O to 2L

Hybridization mix
40% Formamide
5x SSC
1x Denhard's
100ug/ml fish testis DNA
100ug/ml yeast tRNA

NTE
0.5M NaCl
10mM Tris pH7
5mM EDTA

Blocking
1% Boehringer blocking reagent in MABT

MABT pH7.5
100mM maleic acid
150mM NaCl
0.1% Tween 20

100x Levamisole/ Tween-20
50mg/ml Levamisole
10% Tween-20

Synthesis of Labelled RNA probe (Roche)

Linearise DNA
Digest 5ug plasmid O/N in 40ul

Transcription mix
Mix the reagents in the following order
DEPC water
10x Transcription buffer  2ul
Nucleotide mix (DIG or FLUORESCEIN)  2ul
Linearised plasmid  1ug
RNase Inhibitor (40U/ul)  1ul
SP6, T7 or T3 polymerase (10U/ul)  1ul
Total  20ul

Incubate 37C 2h
Run 1ul on agarose gel with linearised plasmid to see if probe is synthesized, run fast <15min. An RNA band, 10 fold more intense than the plasmid should be seen.

Precipitation
For DIG-labelled probe:
TE (50mM TrisHCl, 1mM EDTA, pH8.0)  100ul
4M LiCl  10ul
Ethanol  300ul

For FLUORESCEIN-labelled probe:
DEPC H2O  100ul
Isopropanol  300ul
5M NH4 acetate  78ul

Mix and precipitate at -20C 1h-O/N
Spin 10min max 4C
Remove supernatant
Add 70% EtOH
Spin 5min 4C
Wash again with 70% EtOH
Air dry
Resuspend in 50ul DEPC H2O
Spec concentration

Store -80C

10x transcription buffer
40mM Tris HCl pH8.25
60mM MgCl2
20mM Spermidine

Nucleotide mix
10mM GTP
10mM ATP
10mM CTP
6.5mM UTP
3.5 mM DIG or FLUO-UTP pH8

A regulatory ribosome

Isn't it exciting? To think that ribosomes might have specialized activities, that there might be different ribosome populations translating different subsets of mRNAs. I'm suddenly reminded at how exciting this is. How revolutionary this thinking is..