|
| Status |
Public on Apr 03, 2018 |
| Title |
Max at 100 nM concentration |
| Sample type |
protein |
| |
|
| Source name |
Human Max
|
| Organism |
Homo sapiens |
| Characteristics |
transcription factor: Max
protein concentration: Protein concentration: 100nM
|
| Growth protocol |
Plasmids were transformed into BL21 (DE3) expression strain of E. coli (New England BioLabs). Overnight bacterial culture (5 mL) was diluted into 1 L LB (10 g BACTO Tryptone (BD); 5 g BACTO Yeast Extract (BD); 10 g NaCl; pH 7.5) and grown to OD 0.6 (600 nm visible light). Protein expression was then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hours at 37ºC. Cells were pelleted by centrifugation @ 12,000 rpm for 10 min and stored at -20ºC. Each pellet (from 1 L culture) was thawed in 15 ml lysis buffer (150 mM Tris-HCl, pH 8.0; 150 mM NaCl; 2 mM dithiothreitol (DTT); 1 tablet complete Mini, EDTA-free protease inhibitor cocktail (Roche, #11 836 170 001); 50 uM C4H10O6Zn (zinc acetate); 1 mg/ml chicken egg white lysozyme (Sigma, # L6876)) and subjected to two freeze-thaw cycles in dry ice/ethanol. To digest DNA, to each 15 ml lysis solution was added 10 uL recombinant, RNase-free DNAse I (Roche, #04 716 728 001, 10 U/ul), 30 uL 1 M MgCl2, and 2 mL 10% Triton X-100, and digested at room temperature until solution was runny (~1 hr). Solutions were centrifuged at 12,000 rpm for 25 min at 4ºC, and supernatant was used for protein purification.
|
| Extracted molecule |
protein |
| Extraction protocol |
Full-length open reading frames were cloned into the Gateway pDEST17 (N-terminal His-tag) expression vector by recombinational cloning from previously created pENTR clones. All proteins were produced by over-expression in E. coli BL21 (DE3) cells (New England BioLabs), and purified by FPLC (AKTAprime plus) using HisTrap FF affinity columns (GE Healthcare). Anti-His Western blots were performed to assess protein quality and concentration.
|
| Label |
Alexa 488
|
| Label protocol |
Proteins were tagged with N-terminal His by cloning. Protein-bound arrays were incubated with Alexa-488-conjugated rabbit polyclonal antibody to His (Invitrogen).
|
| |
|
| Hybridization protocol |
Double-stranded microarrays were first pre-moistened in PBS / 0.01% Triton X-100 for 5 min and blocked with PBS / 2% (wt/vol) nonfat dried milk (Sigma) for 1 h. Microarrays were then washed once with PBS / 0.1% (vol/vol) Tween-20 for 5 min and once with PBS / 0.01% Triton X-100 for 2 min. Proteins were diluted to 200 or 100 nM in a 175-μl protein binding reaction containing PBS / 2% (wt/vol) milk / 51.3 ng/μl salmon testes DNA (Sigma) / 0.2 μg/μl bovine serum albumin (New England Biolabs). Preincubated protein binding mixtures were applied to individual chambers of a four-chamber gasket cover slip in a steel hybridization chamber (Agilent), and the assembled microarrays were incubated for 1 h at room temperature. Microarrays were again washed once with PBS / 0.5% (vol/vol) Tween-20 for 3 min, and then once with PBS / 0.01% Triton X-100 for 2 min. Alexa-488-conjugated rabbit polyclonal antibody to His (Invitrogen) was diluted to 50 μg/ml in PBS / 2% milk and applied to a single-chamber gasket cover slip (Agilent), and the assembled microarrays were again incubated for 1 h at 20°C. Finally, microarrays were washed twice with PBS / 0.05% (vol/vol) Tween-20 for 3 min each, and once in PBS for 2 min. After each hour-long incubation step, microarrays and cover slips were disassembled in a staining dish filled with 500 ml of the first wash solution. All washes were performed in Coplin jars on an orbital shaker at 125 r.p.m. Immediately following each series of washes, microarrays were rinsed in PBS (slowly removed over approximately 10 seconds) to ensure removal of detergent and uniform drying.
|
| Scan protocol |
Protein-bound microarrays were scanned to detect Alexa-488-conjugated antibody (488 nm ex, 522 nm em) using at least three different laser power settings to best capture a broad range of signal intensities and ensure signal intensities below saturation for all spots. Microarray TIF images were analyzed using GenePix Pro version 6.0 software (Molecular Devices), bad spots were manually flagged and removed, and data from multiple Alexa 488 scans of the same slide were combined using masliner (MicroArray LINEar Regression) software.
|
| Description |
same as raw data file from GSE59845, sample GSM1447392
|
| Data processing |
To correct for any possible non-uniformities in protein binding, we adjusted the Alexa 488 signals according to their positions on the microarray. We calculated the median normalized intensity of the 15 x 15 block centered on each spot and divided the spot's signal by the ratio of the median within the block to the median over the entire chamber. For each unique sequence represented on the array, we then calculated the median over replicate spots, applying a natural logarithm transformation, and then normalized the signal to the [0,1] range.
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| |
|
| Submission date |
Apr 17, 2017 |
| Last update date |
Apr 04, 2018 |
| Contact name |
Raluca Gordan |
| E-mail(s) |
raluca.gordan@duke.edu
|
| Organization name |
Duke University
|
| Department |
Center for Genomic and Computational Biology
|
| Street address |
101 Science Dr, CIEMAS 2179
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
| |
|
| Platform ID |
GPL17173 |
| Series (2) |
| GSE97794 |
Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding |
| GSE97885 |
Divergence in DNA specificity among paralogous transcription factors contributes to their differential in vivo binding [RG_MycMaxMad_v1] |
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