User:Karmella Haynes/Notebook/Polycomb project/2010/09/15

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

09/15/10

  • ✓ Western: KAH130-4 IP samples
  • ✓ RT-PCR: test new primers for Nanostring gene candidates
  • ✓ Cell culture: thaw HEK Gal4-EED line (from Mirra)
  • ✓ ChIP: IP for KAH126-1, 130-2 (double x-linked), and 130-4 (single x-linked) for DNA isolation
  • ✓ RT-PCR: Nanostring gene candidates IL4, MMP12, THPO, TNF on full cDNA set



Western
> IP samples from 9/13/10; already prepped for loading (2 wells worth)
> Prep input and sup samples: 22.5 protein + 7.5 4x loading dye
--> 4x loading dye (Invitrogen) w/ freshly added DTT (200 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.

Gels 1 & 2

1. PageRuler Plus (10 μL)
2. KAH130-4 input
3. KAH130-4 α-myc sup
4. KAH130-4 α-myc IP
5. KAH130-4 α-IgG sup (neg ctrl)
6. KAH130-4 α-IgG IP (neg ctrl)
7. PageRuler Plus (10 μL)
8. 1x dye 9. 1x dye 10. 1x dye

File:KAH 091510 ps1.tif
Ponceau S stained filters

> Block: 5% milk/PBST, R.T./1 hr.

> Primary staining: 5% BSA/PBST, 4°C/o.n.

  1. rabbit α-DsRed 632496, 1:1000, 5 mL
  2. rabbit α-H3K27me3 07-449, 1:500, 5 mL


8/28/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.

  1. donkey α-rabbit-HRP, 1:5000; 5 mL
  2. donkey α-rabbit-HRP, 1:5000; 5 mL

> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)

  • KAH130-4: 79 kD
  • H3K27me3: 15 - 17 kD


Western blot 9/16/10
Conclusions:

  • Blot 1: 130-4 pull-down looks perfect. Very clean and specific.
  • Blot 2: 17 kD H3K27me3 shows up at right size in input and sup lanes, but what is the mysterious 28 kD band? Same as observed before with DMA/formaldehyde cross-linked KAH126-1 & 132-8 (8/28/10). Could be something coming of of the α-myc beads during elution that is recognized by α-H3K27me3 primary or α-rabbit secondary. No 17 kD band. Try total IP next time (and myc-bead/ no chrom control?).



RT-PCR
> Use cDNA from Nanostring set
> Primers + NS Templates:

  1. HHEX 28C (126 bp) + KAH129-4 -/+ dox (2 rxns)
  2. IL4 30A (107 bp) + KAH129-4 -/+ dox (2 rxns)
  3. IL4 30B (89 bp) + KAH129-4 -/+ dox (2 rxns)
  4. MMP12 31A (127 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox, KAH129-4 -/+ dox (6 rxns)
  5. MMP12 31b (99 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox, KAH129-4 -/+ dox (6 rxns)
  6. NPPA 32A (99 bp) + KAH126-1 -/+ dox (2 rxns)
  7. NPPA 32B (99 bp) + KAH126-1 -/+ dox (2 rxns)
  8. THPO 33A (103 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox (4 rxns)
  9. THPO 33B (105 bp) + KAH126-1 -/+ dox, KAH128-8 -/+ dox (4 rxns)
  10. TNF 34A (119 bp) + KAH128-8 -/+ dox (2 rxns)
  11. TNF 34B (107 bp) + KAH128-8 -/+ dox (2 rxns)


Reagent Single rxn
cDNA 0.5 (1:1 cDNA)
10 μM primers 1.0
2x GoTaq Green 10.0
dH2O 8.5
  20.0 μL


--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

File:KAH 091510 gel1.tif

> Conclusions:

  • MMP12, THPO, and TNF successfully detected. Repeat PCR for whole cDNA set, use fewer cycles (32, same as p16).
  • IL4 worked somewhat; a little non-specific. Try fewer cycles.
  • HHEX shows very faint bands, but product appears to be the right size.
  • NPPA failed. Order new primers.



RT-PCR
> Use cDNA from Nanostring set (omit KAH158, 159)
> Primers + NS Templates:

  1. IL4 30B (89 bp) + NS cDNA (16 rxns)
  2. MMP12 31B (99 bp) + NS cDNA (16 rxns)
  3. THPO 33A (103 bp) + NS cDNA (16 rxns)
  4. TNF 34A (119 bp) + NS cDNA (16 rxns)


Reagent Single rxn MM (x17)
cDNA 0.5 (1:1 cDNA) ---
10 μM primers 1.0 17.0
2x GoTaq Green 10.0 170.0
dH2O 8.5 144.5
  20.0 μL


--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
  • 72°C/ 3 min.
  • 4°C/ ∞

File:KAH 091510 gel2.tif

> Conclusions:

  • IL4 too non-specific. Do not see induction in specifically in 129-4
  • MMP12 looks good, but has ns lower band. Try again with 1:10 dilution of cDNA and primer pair 31A
  • THPO, hard to see differences. Try again with 1:10 dilution of cDNA
  • TNF looks great. Use data for figure.
  • Try the successful primers on previous biological replicates.



ChIP: myc-bead pull-down
> See 6/29/10
> Sonicated ~6 mL samples previously prepped according to Qingqing's protocol
--> Add:

  • 720 μL 10% Triton X-100
  • 72 μL 10% Na-deoxycholate (DOC)
  • 500 μL TE (pH 8.0)
  • 6 μL 1000x PLAAC
  • 60 μL 100x PMSF


> Binding:

  1. KAH126-1: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  2. KAH126-1: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
  3. KAH132-8: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  4. KAH132-8: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
  5. KAH130-4: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  6. KAH130-4: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

--> Rotate at 4°C overnight
--> Wash and elute tomorrow


9/14/10
> Wash & elute IP's (Keep everything on ice)
--> Prepare washing buffer (complete sonication buffer):

  • 6 mL Buffer III
  • 60 μL 100x PMSF
  • 6 μL 1000x PLAAC
  • 720 μL 10% Triton X-100
  • 72 μL 10% Na-deoxycholate (DOC)
  • 500 μL TE (pH 8.0)

--> Spin down beads at 3000 rpm/ 3 min./ 4°C
--> Save 100 μL Supernatant. Discard remaining sup.
--> Wash beads in 500 μL wash buffer (1 min.). Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. Repeat three more times (4 washes total)
--> Add 500 μL 1% SDS/TE buffer. Incubate at 65°C/ 15 min. Vortex every 2 min.
--> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube.


> Reverse crosslinks and digest protein
--> Add 400 uL 1% SDS/TE buffer to 100 uL saved Supernatant.
--> Thaw Input aliquot for each cell line. Add 400 uL 1% SDS/TE buffer to 100 uL Input.
--> Samples:

  1. KAH126-1 input
  2. KAH126-1 α-myc Sup
  3. KAH126-1 α-myc IP
  4. KAH126-1 α-IgG Sup
  5. KAH126-1 α-IgG IP
  6. KAH130-4 input
  7. KAH130-4 α-myc Sup
  8. KAH130-4 α-myc IP
  9. KAH130-4 α-IgG Sup
  10. KAH130-4 α-IgG IP
  11. KAH132-8 input
  12. KAH132-8 α-myc Sup
  13. KAH132-8 α-myc IP
  14. KAH132-8 α-IgG Sup
  15. KAH132-8 α-IgG IP

--> Add 20 μL (20 μg) Pronase to each sample
--> Incubate at 42°C/ 1 hr. --> Incubate at 65 °C overnight