User:Celina Demers/Notebook/Labwiki1/2010/08/28

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Example 1
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Objectif
Western blot to confirm the interaction between Mll with Ash2l,P45 and others

Related experience or papers
// leave a comment about a paper here
 * 1) Paper1 pmid=17707229

Protocol
Cell Culture, Immunoprecipitation, and Quantitative Mass Spectrometry MEL (clone 745) and CB3 cells were cultured in c. An expression vector encoding NF-E2/p45 (Kotkow and Orkin, 1995 K.J. Kotkow and S.H. Orkin, Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer, Mol. Cell. Biol. 15 (1995), pp. 4640–4647. View Record in Scopus | Cited By in Scopus (129)Kotkow and Orkin, 1995) was stably transfected into the CB3 cell line, and clones expressing NF-E2/p45 were selected in 1 mg/ml G418. CB3 and CB3+p45 clones were induced to differentiate along the erythroid lineage by adding 1.5% final DMSO for 3 days. MEL cells were induced to differentiate along the erythroid lineage by adding 2% final DMSO for 3 days and nuclear extracts were prepared as previously described (Brand et al., 2004). Anti-NF-E2/p45, -ASH2L, and -MLL2 IPs were performed as described (Brand et al., 2004) with the following modifications. Abs crosslinked to Dynabeads-protein A (Dynal) were used for IP. Elution of the proteins was performed at 37°C in 50% acetonitrile containing 0.1% TFA. Eluted proteins were labeled with the isotopically labeled ICAT reagents and identified and relatively quantified by mass spectrometry as described previously (Ranish et al., 2007). The probability of identification was determined for each protein using the PeptideProphet and ProteinProphet algorithms (Nesvizhskii et al., 2003).

Chromatin Immunoprecipitation ChIP assays were performed as previously described (Brand et al., 2004) except that sonication was done with a Bioruptor (Diagenode). Real-time qPCR analysis was done on a ABI Prism 7500 detector with Taqman probes and primers (sequences available in the Supplemental Data). To calculate the enrichment of each protein to a particular target DNA, values obtained (via the standard curve method) for each target were divided by the amount of the corresponding target in the input fraction. Enrichments obtained from mock IPs performed in parallel with normal IgG were then subtracted from the enrichment values obtained with specific Abs. All the enrichments are expressed as a function of the highest enrichment obtained on the locus (set to 1).

Methyltransferase Assays NF-E2/p45-, Mock-, or MLL2-associated proteins were isolated on beads and incubated with 1 mg/ml purified core histones (Roche) or nucleosomes (prepared as described in Brand et al., 1999) for 1 hr at 30°C in the presence of 6.5 μM of [3H]s-adenosyl methionine in the EX50 buffer (10 mM HEPES K+ [pH 7.6], 50 mM KCl, 10% Glycerol, 1.5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT, protease inhibitor cocktail). Proteins were eluted by adding ¼ volume of 4× loading dye buffer (100 mM Tris [pH 6.8], 40% glycerol, 8% SDS, 286 mM DTT, 0.4% bromophenol blue) and incubating at 95°C for 5 min. Proteins were separated by SDS-PAGE and electrotransferred to PVDF membrane before being revealed by Ponceau red and film exposure. Alternatively, eluted proteins were spotted onto phosphocellulose P81 squares and the radioactivity was counted in the presence of scintillation cocktail after washing the squares 4 times in 50 mM Na2HPO4 (pH 9.0).

ASH2L and MLL2 Knockdowns in MEL Cells Stable MEL cell lines stably expressing anti-ASH2L or anti-MLL2 shRNA in a doxycycline (Dox)-dependent manner were established as described in Supplemental Data. The knockdowns were induced by adding 5 μg/ml final of Dox in the culture medium for 3–4 days. Transcription of the βmaj-globin gene was assessed by RT-qPCR with Taqman probes (sequences available in Supplemental Data) after RNA extraction and RNase-free DNase I digestion (QIAGEN RNAeasy kit).

Material used
(this is just an example)
 * RPMI,serum,pen/strep
 * Dynabeads
 * Mll 2 antibody from Millipore cat#0001 lot#A-1

Method
No changes

Discustion
Figure 1. NF-E2 Associates with the H3K4 Methyltransferase Complex MLL2 in Differentiated Erythroid Nuclear Extracts(A) Western blot analysis of proteins immunoprecipitated from an erythroid nuclear extract via Abs against MLL2 and NF-E2/p45. A mock IP with normal rabbit IgG was used as a negative control. Abs used for western blot are indicated on the right. Asterisk indicates Ab heavy chain.(B) Western blot analysis of proteins immunoprecipitated from an erythroid nuclear extract via Abs against ASH2L. Asterisk indicates Ab heavy chain.(C) NF-E2/p45 and mock immunoprecipitated proteins were used in an in vitro methylation assay in the presence of [3H]SAM and equal amounts of purified core histones (left) or nucleosomes (right) as a substrate. The top panel is an autoradiograph of the bottom panel (stained with Ponceau red).(D) 11 amino acid peptides corresponding to the N-term part of histone H3 that are either wild-type (WT), acetylated at lysine 9 (AcK9), mutated at position 9 (K/A9), or mutated at lysine 9 and trimethylated at lysine 4 (K4me3-K/A9) were used as a substrate for methylation by NF-E2/p45-, mock-, or MLL2- associated proteins and the radioactivity was counted (in cpm) after a filter binding assay. Error bars correspond to the standard deviation of 3 counts of 1 min each. The experiment was performed at least twice independently.

Conclusion
Send to molecular cell for publication

Next Experiment
User:Celina Demers/Notebook/Labwiki1/2010/08/27


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