|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
ATF2 Antibody detects endogenous levels of total ATF2.
Activating transcription factor 2 (ATF2; also known as cAMP-dependent transcription factor ATF-2) has oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin tumors and breast cancer. ATF2 requires phosphorylation by Jun N-terminal kinase (JNK), p38 (MAPK14), or extracellular-signal-regulated kinase 1 (ERK1) in order to be transcriptionally active. After its activation following stress and cytokine stimuli, ATF2 contributes to the cellular responses to hypoxic or osmotic stress, DNA damage, viral infection and cell death. The precise transcriptional output of ATF2 is dictated by its dimerization partners, which are predominantly members of the AP-1 family. ATF2 homodimers display poor transcriptional activity, and thus heterodimerization of ATF2 is essential for its transcriptional function. Depending on the heterodimeric partner, ATF2 binds to different response elements on target genes and elicits distinct transcriptional programs. In unstimulated conditions, ATF2 is maintained in a transcriptionally inactive form by intramolecular binding of its C-terminal DNA-binding domain to its N-terminal activation domain. Binding of transcriptional co-activator and histone acetyltransferase CBP [CREB (CRE-element-binding protein)-binding protein] or the adenovirus 13S E1A protein to the bZIP region alleviates the intramolecular inhibition and leads to activation of transcription. ATF2 also interacts with the transcriptional co-activator p300. This complex formation is important for the regulation of c-Jun gene expression in response to inducers of cell differentiation such as retinoid acid or adenovirus E1A in mouse embryonal carcinoma cells. In contrast, interaction with the ATP-dependent helicase TIP49b results in the down-regulation of ATF2-dependent transcription activity.