10/06/10
- ✓ Order oligos: new primers for MMP12 and TNF ChIP-qPCR
- ✓ Cell culture: split HEK Gal4-EED's; set up dox-induced Gal4-EED time course plate (12-well, ~50,000 cells/well)
- ✓ ChIP/ co-IP: Protein A Sepahrose-H3K27me3 IP
Oligos
- MMP12 C f2; gaattgcaagcagtggtgctg
- MMP12 C r2; cagaccgtccccatacaatc
- MMP12 C f3; gcagattcttgctgatctgttc
- MMP12 C r3; ctcacagcagaataaccagc
- MMP12 D f1; cataacagtccttcaaaacc
- MMP12 D r1; tggtctaggaggctgtctgc
- MMP12 D f2; ctgagatgactgagggtaaag
- MMP12 D r2; ggagttcacttctgttatgg
- TNF C f2; gagaagcaactacagacc
- TNF C r2; cgtgggtcagtatgtgagag
- TNF C f3; ccagatgagctcatgggtttc
- TNF C r3; gctgggtgtgccaacaactg
- TNF D f1; gtgaaagatgtgcgctgatag
- TNF D r1; cagacatcctgtctctccatc
- TNF D f2; catggaaggtgctcactaag
- TNF D r2; ccacatctgtctccatatc
ChIP co-IP optimization
> Not much luck with endogenous protein IP using Qingqing's protocol
> Try simplified approach:
--> Bind antibody with chromatin overnight
--> Bind Protein A* beads with complexes for 5 hours
Note: *H3K27me3 ab 07-449 was Protein A purified (Millipore)
--> Wash with complete sonication buffer (same as IP using myc-conjugated beads)
--> Elute with Western loading buffer
Today:
> Try...
- chromatin + antibody, then beads and
- beads + antibody, then chromatin
> Prepare complete sonication buffer (see ###)
> Prep 200 μL Protein A sepharose by washing and resuspending (3 times) in complete sonication buffer
> Antibody binding
--> Use H3K27me3 07-449
--> Use double x-linked KAH126-1 (surplus chromatin, not important)
| Reagent |
1 |
2 |
3 |
4
|
| α-H3K27me3 07-449 |
2.0 |
--- |
2.0 |
---
|
| rabbit IgG ab |
--- |
2.0 |
--- |
2.0
|
| KAH126-1 dx |
250.0 |
250.0 |
--- |
---
|
| Protein A beads |
--- |
--- |
10.0 |
10.0
|
| Sonication buffer |
250.0 |
250.0 |
500.0 |
500.0
|
--> Rotate at 4°C/ overnight
|
Project name
Main project page
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