User:Karmella Haynes/Notebook/Polycomb project/2010/08/27

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08/27/10

  • ✓ Western blot: ChIP/ Co-IP
  • ✓ Microscopy: secondary staining and mounting (see 8/26/10)



Western blot: ChIP/ Co-IP
> IP samples from 8/20/10; 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 (100 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.

Gels 1 & 2

1. PageRuler Plus (10 μL)
2. KAH126-1 input
3. KAH126-1 α-myc sup
4. KAH126-1 α-myc IP
5. KAH126-1 α-IgG IP (neg ctrl)
6. KAH132-8 input
7. KAH132-8 α-myc sup
8. KAH132-8 α-myc IP
9. KAH132-8 α-IgG IP (neg ctrl)
10. PageRuler Plus (10 μL)

Note: In gel #2, samples misloaded as:
1, 4, 5, 2, 3, 6...10

File:KAH 082710 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, mouse α-GAPDH-HRP 1:5000; 5 mL

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

  • KAH126-1: 43 kD
  • KAH132-8: 35 kD
  • H3K27me3: 15 - 17 kD


File:KAH 082810 WB1.tif
Conclusions:

  • KAH126 pull-down was successful and specific for α-myc beads (top); no signal for KAH132 (?)
  • Both KAH126 and 132 appear to Co-IP with 28 kD band in H3K27me3 staining; no idea what this is (di-nucleosomes?)
  • Perhaps DMA/ formaldehyde is too much crosslinking, given that Pc-ATFs are over-expressed and concentrated in the nucleus
  • Try new preps, using formaldehyde x-linking only