5. Materials and Methods
5.8 Electrophoretic Mobility Shift Assay
BB trace) was added to the beads and samples were heated to 95 °C for 5 min, briefly cooled on ice and spinned at maximum speed for 15 seconds. Supernatants, avoiding beads-carrying over, were subjected to SDS (8%) gel electrophoresis followed by semi-dry electroblotting (100 V, maximum mA, maximum Watts) for 1,5 h. Membrane used was from GE Healthcare (nitrocellulose Hybond ECL). Blot was briefly washed in TBS-Tw (TBS, 0.2% Tween), blocked in 3% nonfat dry milk plus 2% BSA in TBS-Tw, and primary antibody was applied o/n @4°C (a-Cnc 1:5000, a-Col 1:3000), followed by TBS-T washings (4x), 2nd antibody application (a-mouse- or a-rb-HRP at 1:10000, 1 h rt in TBS-T, 3% BSA) and HRP detection using a Lumiglo substrate (Cell Signaling Technology, Inc).
5.7 In vitro protein expression
After cloning of the open reading frames – encoding cDNA sequences of transcription factors of interest in the pTNT vector (§5.2), recombinant plasmid DNA was EtOH-precipitated (1/10 V NaAc 3M pH 5.3) followed by three 70% EtOH washing steps. 8.5 µg of plasmid DNA was used as a template for in vitro transcription and translation using the TNT wheat germ cell SP6 system from Promega. Reaction was at 25 °C for 2 h, then put on ice and subsequently stored at -20 °C.
Mock reactions were performed by using as template the same amount of pTNT plasmid DNA. To check efficiency of the in vitro protein expression 1 µl of the reaction was loaded on 8% SDS gel followed by immunoblotting using the appropriate antibody. 0.5-4.5 µl of the TNT expressed protein factor was used in DNA-protein interaction assays (§5.8, §5.9).
5.8 Electrophoretic Mobility Shift Assay 5.8.1 Preparation of labeled DNA fragment
DNA oligos for upper and lower strand spanning the recognition site to be tested were ordered from Operon as HPLC purified. Sequences of the complementary oligos partially overlap leaving 3’
overhangs of 3-8 nucleotides in the double stranded form to be filled in by the 5’-3’ Klenow enzyme activity.
Oligo stocks were dissolved to 100 pmoles/ µl (100 µM stock concentration) in ddsH2O (mQ).
Then 5 µg of each oligo strand were mixed in 100 µl TE; 150 mM NaCl. Solution was mixed by vortexing, spinned-down and placed at 94 °C for one minute. Heat-block was turned off and left to reach room temperature (~8 hours) to allow for perfect annealing of the complementary strands.
Concentration of the double-stranded oligo form is 100 ng/µl.
200-300 ng of the ds-oligo form were used in the Klenow labeling reaction. Klenow enzyme used was from Biolabs (3’-5’ exo-) (New England BioLabs, NEB). Isotope (α-32P dCTP) was ordered from Hartman Analytic, Braunschweig. For Klenow labeling reaction of ds-oligos to be used in EMSAs the isotope used was of specific activity 800Ci/mmol.
Vfinal 20 µl Klenow labeling reaction
2µl NEB2 buffer (NEB)
2 µl d[AGT]P 2.5 mM each => final c= 0.25 mM 200-300 ng of ds-oligo with 3’ overhangs
1 µl Klenow (3’-5’ exo-) 5 units (NEB) 1 µl α-32P dCTP 10 µCi
Reaction was set on ice, enzyme was added last and incubation was at room temperature (22 °C) for 45 min. At the end of the incubation 30 µl of TE was added and probe was passed through G25 column (GE Healthcare) to eliminate presence of unincorporated nucleotides. 1 µl of the probe was brought juxtaposed to a Geiger-Müller detector (Type EP15, Mini 900 Series, Thermo Scientific) (Perspective Instruments Ltd, UK) to assess counts per second (cps)/µl. By using fresh isotope (less than one half-time) and 200-300 ng of template the probe generated was 500-1000 cps/µl. A dilution in TE was prepared to adjust counts to 150 cps/µl and from this 1 µl was used in the EMSA reactions. In the final dilution the molecular concentration of the probe was also calculated (that was used to assess the amount of cold oligo competitors at the designated molar excess). Probes generated the above way were used at a concentration of ~0.05-0.2 pmoles in the EMSA reaction.
150-200 cps enables signal detection using a Typhoon 9400 scanner (Amersham Biosciences; Image Quant 5.0 Software) after three hours of exposure at room temperature on a Phosphoimager Screen (Amersham Biosciences) or for autoradiography on X-ray film after over-night exposure at -80 °C.
5.8.2 EMSA – Binding reaction
EMSA reactions were set on ice in a final volume of 20µl including 1x Binding Buffer (10mM Hepes pH 7.9, 60mM KCl, 8.4 % glycerol, 1mM EDTA, 2.5mM MgCl2, 1mM DTT, 0.2µM ZnAc).
polydIdC was added to a concentration 50-100 ng/µl and BSA (optional, MB grade, helps stabilize complex formation, helpful in super-shift reactions) to a final concentration 50-200 ng/µl. Protein factor was added (0.5-5 µl of TNT expressed protein or crude nuclear extract ~20 µg) and equilibrated in the binding buffer in the presence of dIdC for 10-15 min @ rt. Then the probe was added (150-200 cps) and binding reactions were incubated at room temperature for 30 min. For super-shift reactions antibody (0.5-1 µl anti-Col) and protein factor were preincubated for 20 min at rt and then probe was added. Binding reactions with crude nuclear extract were performed on ice for 45 minutes to minimize nuclease activity that degrades probe, in the presence of 100-200 ng polydIdC. MgCl2 was also omitted (included in nuclear extraction buffer). [Embryonic crude nuclear extracts were prepared essentially as described in Sullivan et al, 2000 Drosophila Protocols, CSHL Press, Chapter 31, p. 553-557.]
In the meanwhile the native gel was prerun at 4 °C at 120 V-10 mA to equilibrate in buffer conditions and minimize presence of Aps and Temed. Pre-ran buffer was exchanged with
fresh buffer (0.25xTBE pH 8.3, 0.1% glycerol) before loading the samples. Native gel composition was 6% acrylamide 59:1, 0.25xTBE pH 8.3, 2.5% glycerol (7.5% APS, 0.04% Temed). Reactions were run for 1,5-2 h at 160V, 12 mA. (Bromophenol blue buffer consisting of 10mM Tris pH 8.0, 1 mM EDTA ,10% glycerol, loaded at a separated lane or added in the free-probe reaction reaches at 2/3 gel distance). Then the gel was dried on 3MM Whatman paper under vacuum (80°C) and exposed.
Oligos used for mobility shift assays; after annealing either the 3’ overhangs (underlined) were filled in by Klenow emzymic activity to generate labeled probe or used as competitors in 50-150x molar excess.
Name Sequence Len Purpose
TCFsite_HMG GGTCCGGATCAAAAGGAGCCCGGTTGA 27
TCFsite_HMG_Rev AACATTTGTCAACCGGGCTCCTTTTGAT 28
§2.5.2
TCFsite_optimal GGTCCGGATCAAAGGAGCCCGGTTGA 26
TCFsite_optimal_Rev AACATTTGTCAACCGGGCTCCTTTGAT 27
TCFsite_compet GGTCCGGATCAAAGGAGCCC
TCFsite_competR GGGCTCCTTTTGATCCGGACC
HMG_compet GCCCGGTTGACAAATGTT
HMG_competR AACATTTGTCAACCGGGC
Colsite_F TTTCGGCAGCAATTCCCCAATGGCATTT 28 §2.7.2
Colsite_R TAAGTGAAATGCCATTGGGGAATTGCTG 28
Colsite_optimal TTTCGGCAGCAATTCCCCAAGGGCTTTT Colsite_optimalRev TAAGTGAAAAGCCCTTGGGGAATTGCTG
colsite_mut1 TTCGGCAAGGATTCCCCAATGGCATTTCAC
colsite_mut1R GTGAAATGCCATTGGGGAATCCTTGCCGAA
colsite_mut2 TTCGGCAGCAATGAACCAATGGCATTTCAC
colsite_mut2R GTGAAATGCCATTGGTTCATTGCTGCCGAA
oligo3compet ATCCCAATCCCTGGTAGCCGTAAAT 25 -3674_-3650; Fig. 2_46
oligo3competR ATTTACGGCTACCAGGGATTGGGAT 25
oligo3mutcompet ATCCCAACGGCTTTTAGCCGTAAAT oligo3mutcompetR ATTTACGGCTAAAAGCCGTTGGGAT
col_cnc_site TCGGCAGCAATTCCCCAATGGCATTT 26 §2.8.1
col_cnc_siteRev ATCTAAGTGAAATGCCATTGGGGAATTG 28
bZIPsite_F CCATTTGCCTAATTTCTATTTCGG
bZIPsite_R GGAATTGCTGCCGAAATAGAAATTAGG
positive target site for C/EBP binding shift
oligo4_fkhsite_F GTAAATGTCAACATCCATTAGAGA oligo4_fkhsiteR GAGGTCTCTAATGGATGTT
forkhead site in the 4th block matching Slp1 binding site