|
| Status |
Public on Jul 17, 2016 |
| Title |
CLAMP 6 zinc finger (ZF) PBM |
| Sample type |
protein |
| |
|
| Source name |
IVT sample of GST-tagged CLAMP 6-ZF construct
|
| Organism |
Drosophila melanogaster |
| Characteristics |
clamp construct: GST-tagged CLAMP 6-ZF protein
|
| Treatment protocol |
Microarray was incubated with GST-tagged CLAMP 4-ZF sample, or GST-tagged CLAMP 6-ZF sample
|
| Growth protocol |
N/A
|
| Extracted molecule |
protein |
| Extraction protocol |
GST-tagged CLAMP ZF proteins were produced by in vitro transcription translation (IVT) from pDEST15 GST expression plasmics (Invitrogen). IVT was performed using the TnT Quick-coupled Transcription and Translation Systems Kit (Promega) following standard procedure with the one adaption - zinc acetate was added to a final concentration of 50 uM. Proteins were quantified by Western blotting and comparing to a dilution series of GST standard. Concentration of samples used was: CLAMP 4 ZF sample (24.3 nM); CLAMP 6 ZF sample (13.9 nM).
|
| Label |
Alexa488
|
| Label protocol |
Microarray-bound protein was labelled by incubation for 20 min with 0.05 mg/ml Alexa488-conjugated anti-GST antibody (Invitrogen, #A11131).
|
| |
|
| Hybridization protocol |
Protein samples were incubated with microarray for 1 hour @ 200 nM protein concention in binding buffer ( 10 mM Tris-HCl, pH 7.4; 0.2 ug/ul bovine serum albumin (BSA) (New England Biolabs #B9001S); 0.3 ng/ul Salmont testes DNA (Sigma #D7656), 2% non-fat dry milk; 1 mM dithiothreitol (DTT); 1 mM ehtylenediaminetetraacetic acid (EDTA); 50 uM zinc acetate dehydrate; 80 mM NaCl).
|
| Scan protocol |
Microarray scanning performed using GenePix 4400 A (Moleclar Devices)
|
| Description |
DNA binding
|
| Data processing |
Image analysis and data normalization performed using Gene Pix Pro ver 6 and masliner (MicroArray LINear Regression) software
Median fluorescence intensities were calculated over 8 replicate probes for each unique DNA sequence
|
| |
|
| Submission date |
Jun 16, 2016 |
| Last update date |
Jul 18, 2016 |
| Contact name |
Michael Tolstorukov |
| E-mail(s) |
tolstorukov@molbio.mgh.harvard.edu
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Molecular Biology
|
| Street address |
185 Cambridge Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL22023 |
| Series (2) |
| GSE83442 |
CLAMP Protein Binding Microarray (PBM) experiment |
| GSE83444 |
Expansion of GA dinucleotide repeats increases the density of CLAMP binding sites on the X-chromosome to promote Drosophila dosage compensation |
|
