ATF5 Antibody

What is ATF5 and why is it significant in cellular biology?

ATF5 (Activating Transcription Factor 5) is a 30.7 kDa protein belonging to the ATF/CREB transcription factor family. It functions critically in cellular stress responses, particularly during hypoxia and nutrient deprivation . The protein is characterized by a conserved C-terminal leucine zipper domain that facilitates dimerization and DNA binding, while its N-terminal domains exhibit significant variability allowing for diverse regulatory functions .

ATF5 localizes in both cytoplasm and nucleus, functioning as a dimer to bind DNA. It interacts with several proteins including CCND3 and PTP4A1, which are involved in cell cycle regulation and signal transduction pathways . One of its key roles is maintaining mitochondrial homeostasis and regulating mitochondrial quality control, particularly in response to exercise and cellular stress . ATF5 has been implicated in cancer pathogenesis, with altered expression observed in glioblastoma, breast cancer, and pancreatic carcinoma .

How do I select the appropriate ATF5 antibody for my experimental needs?

When selecting an ATF5 antibody, consider these critical factors:

Species reactivity: Determine whether the antibody recognizes ATF5 in your species of interest. Many commercially available antibodies detect human, mouse, and rat ATF5, but cross-reactivity varies significantly .

Application compatibility: Verify the antibody has been validated for your specific application:

• Western blotting (WB)

• Immunoprecipitation (IP)

• Immunofluorescence (IF)

• Immunohistochemistry (IHC)

• ELISA

Clonality considerations:

• Monoclonal antibodies (e.g., E-10 clone) offer consistency across experiments but may recognize only specific epitopes

• Polyclonal antibodies provide broader epitope recognition but potentially more background

Epitope location: Consider whether your experimental conditions might mask or modify the epitope. For studies investigating post-translational modifications like acetylation at K29, ensure the epitope doesn't include or isn't affected by this site .

Validation data: Review immunoblots, IHC images, and immunofluorescence data from manufacturers to assess specificity and expected banding patterns .

What are optimal Western blot conditions for detecting ATF5?

For successful Western blot detection of ATF5, consider the following protocol optimizations:

Sample preparation:

• Use RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if studying phosphorylation states

• For detecting acetylated ATF5, add deacetylase inhibitors like trichostatin A (TSA)

Protein loading and separation:

• Load 20-40 μg of total protein per lane

• Use 10-12% polyacrylamide gels for optimal separation

• Expected MW: ~30.7 kDa, though post-translational modifications may alter migration

Transfer conditions:

• Semi-dry or wet transfer at 100V for 60-90 minutes

• PVDF membranes generally yield better results than nitrocellulose for ATF5

Blocking and antibody incubation:

• Block with 5% non-fat milk or BSA in TBST

• Primary antibody dilutions typically range from 1:500 to 1:2000

• Incubate overnight at 4°C for optimal results

Controls:

• Include positive controls (cells known to express ATF5)

• Use ATF5 knockdown samples as negative controls

• For acetylation studies, compare samples with and without deacetylase inhibitors

Detection considerations:

• HRP-conjugated secondary antibodies with enhanced chemiluminescence detection work well

• For quantitative analysis, consider fluorescent secondary antibodies

How can I optimize immunofluorescence protocols for ATF5 localization studies?

ATF5 exhibits both nuclear and cytoplasmic localization, making correct fixation and permeabilization critical:

Fixation options:

• 4% paraformaldehyde (10-15 minutes at room temperature) preserves morphology

• Methanol fixation (-20°C for 10 minutes) can improve nuclear epitope accessibility

Permeabilization:

• 0.1-0.3% Triton X-100 for PFA-fixed cells (10 minutes)

• Avoid excessive permeabilization which can affect nuclear membrane integrity

Blocking and antibody incubation:

• Block with 5-10% normal serum from secondary antibody host species

• Include 0.1% BSA to reduce non-specific binding

• Primary antibody dilutions typically 1:50-1:200 for immunofluorescence

• Incubate overnight at 4°C in a humidified chamber

Nuclear counterstaining:

• DAPI or Hoechst for nuclear visualization

• Consider double-staining with mitochondrial markers when studying mitochondrial functions

Microscopy settings:

• Capture z-stacks to accurately assess nuclear versus cytoplasmic distribution

• Include single-stained controls for spectral bleed-through correction

Stress response studies:

• Compare localization before and after cellular stressors (e.g., serum deprivation or staurosporine treatment)

• Document time-dependent changes in localization following stress induction

How can ATF5 antibodies be employed to study cancer biology and chemoresistance?

ATF5 antibodies have become valuable tools in cancer research due to ATF5's role in cancer cell survival and chemoresistance:

Expression profiling in tumors:

• IHC analysis has revealed that ATF5 is highly expressed in glioblastomas, breast cancers, and pancreatic carcinomas compared to normal tissues

• Quantitative analysis correlating ATF5 expression with patient outcomes can help establish prognostic value

Mechanisms of chemoresistance:

• Studies in pancreatic cancer cells demonstrated that ATF5 interference enhanced sensitivity to paclitaxel by modulating BCL-2 and BAX expression

• Western blot analysis with ATF5 antibodies can quantify changes in expression following drug treatments

Therapeutic targeting validation:

• ATF5 antibodies can confirm knockdown efficiency in experiments using siRNA or shRNA approaches

• In a study on mammary tumors, ATF5 knockdown resulted in decreased tumor volume and weight, reduced proliferation rate, and diminished migratory potential

Downstream pathway analysis:

• ChIP assays using ATF5 antibodies have identified direct binding to the ARE region of the Egr-1 promoter, revealing a molecular mechanism for ATF5's role in cell proliferation

• Co-immunoprecipitation with ATF5 antibodies has uncovered interactions with p300, revealing regulatory mechanisms involving acetylation

What methods can be used to study ATF5 post-translational modifications?

ATF5 undergoes several post-translational modifications that regulate its function:

Acetylation detection:

• ATF5 is acetylated at lysine-29 (K29) by p300

• Immunoprecipitate ATF5 with a specific antibody, followed by Western blotting with anti-acetyl-lysine antibodies

• Alternatively, use acetylation site-specific antibodies when available

• Treatment with deacetylase inhibitors like trichostatin A (TSA) enhances detection of acetylated ATF5

Phosphorylation studies:

• Use phospho-specific antibodies when available

• Alternatively, perform immunoprecipitation with ATF5 antibodies followed by Western blotting with anti-phospho-serine/threonine/tyrosine antibodies

• Lambda phosphatase treatment can serve as a control to validate phosphorylation signals

Ubiquitination analysis:

• Immunoprecipitate ATF5 under denaturing conditions (to disrupt protein-protein interactions)

• Western blot with anti-ubiquitin antibodies

• Use proteasome inhibitors (MG132) to enhance detection of ubiquitinated forms

Mass spectrometry approaches:

• Immunoprecipitate ATF5 and subject to tryptic digestion

• LC-MS/MS analysis can identify multiple modifications simultaneously

• SILAC approaches can quantify changes in modification states under different conditions

How can I investigate ATF5's role in mitochondrial stress responses?

ATF5 plays a crucial role in mitochondrial quality control, particularly in response to stress:

Mitochondrial unfolded protein response (UPRmt) assessment:

• Use ATF5 antibodies to track ATF5 translocation to mitochondria during stress

• Co-staining with mitochondrial markers can confirm localization

• RNA-seq analysis in ATF5 knockdown cells has revealed that ATF5 regulates genes involved in the UPRmt pathway

Functional mitochondrial assays:

• Studies have shown that ATF5 knockdown decreases mitochondrial membrane potential and ATP levels

• Measure these parameters using fluorescent dyes (JC-1, TMRE) for membrane potential and luciferase-based assays for ATP

Mitochondrial dynamics:

• ATF5 knockdown impacts mitochondrial fusion and fission by regulating genes like Drp1, Fis-1, Opa1, Mfn1, and Mfn2

• Use immunofluorescence with ATF5 antibodies alongside mitochondrial morphology assessments

Autophagy and mitophagy:

• ATF5 knockdown increases autophagy as measured by LC3B immunofluorescence

• Use ATF5 antibodies in combination with autophagy markers to assess the relationship between ATF5 and mitochondrial clearance

Why might I observe multiple bands when performing Western blot with ATF5 antibodies?

Multiple bands in ATF5 Western blots can occur for several legitimate reasons:

Post-translational modifications:

• Acetylated ATF5 (particularly at K29) may show altered migration

• Phosphorylated forms can appear as higher molecular weight bands

• Ubiquitinated ATF5 will appear as a ladder of higher molecular weight bands

Isoforms and splice variants:

• Different ATF5 isoforms may be expressed in certain tissues or under specific conditions

• Verify these are legitimate isoforms by comparing with literature reports

Degradation products:

• C-terminal fragments may be detected if the antibody recognizes this region

• Improve sample preparation by adding protease inhibitors and keeping samples cold

Cross-reactivity:

• Some antibodies may detect related ATF family members (particularly ATF4, an important paralog)

• Use ATF5 knockout or knockdown samples as negative controls to confirm specificity

Technical suggestions:

• Run a gradient gel to better separate closely migrating bands

• Perform peptide competition assays to identify specific versus non-specific bands

• Pre-adsorb antibodies with recombinant ATF5 protein to reduce non-specific binding

What controls are essential when studying ATF5 function through knockdown or overexpression?

Rigorous controls are critical for accurate interpretation of ATF5 functional studies:

Knockdown experiments:

• Include multiple siRNA/shRNA sequences targeting different regions of ATF5 to rule out off-target effects

• Confirm knockdown efficiency at both mRNA (qRT-PCR) and protein levels (Western blot)

• Include a non-targeting siRNA/shRNA control with similar chemical modifications

• In the study by Frontiers in Endocrinology, researchers systematically validated their knockdown approach before investigating tumor volume effects

Overexpression experiments:

• Include empty vector controls processed identically to ATF5-expressing samples

• Consider using an inducible expression system to control expression levels

• Tag location (N- vs. C-terminal) may affect function; validate function using known ATF5 activities

Rescue experiments:

• Express siRNA/shRNA-resistant ATF5 to demonstrate specificity of observed phenotypes

• Include non-functional ATF5 mutants (e.g., K29R mutant that cannot be acetylated) as negative controls

Functional validation:

• Assess known ATF5 transcriptional targets like Egr-1 using reporter assays

• Measure changes in mitochondrial parameters known to be affected by ATF5 modulation

• Document changes in downstream protein expression (e.g., BCL-2, BAX)

How can I analyze conflicting data regarding ATF5 function in different cellular contexts?

ATF5 exhibits context-dependent functions that may appear contradictory:

Cell type considerations:

• ATF5 is essential for survival in cancer cells but dispensable in non-transformed cells

• In olfactory sensory neurons, ATF5 is required for terminal differentiation and survival

• Document the baseline expression levels of ATF5 and its binding partners in your model system

Stress conditions:

• ATF5 response may differ dramatically between basal and stress conditions

• The physical association between ATF5 and p300 is disrupted after serum deprivation or staurosporine treatment

• Design experiments that explicitly compare multiple stress conditions with appropriate controls

Compensatory mechanisms:

• Absence of ATF5 triggers compensatory increases in PGC-1α and ATF4 expression

• Consider analyzing related family members and pathway components when manipulating ATF5

Technical reconciliation approaches:

• Create a systematic comparison table of experimental conditions across studies

• Perform time-course experiments to capture dynamic responses

• Use genetic approaches (CRISPR/Cas9) for complete knockout versus transient knockdown to distinguish acute versus chronic effects

Integrated analysis framework:

• Consider multiple functional outputs simultaneously (e.g., proliferation, survival, mitochondrial function)

• Use systems biology approaches to model ATF5 in its network context

• When possible, validate in vitro findings with in vivo models to assess physiological relevance

How can ATF5 antibodies be utilized in studying mitochondrial quality control mechanisms?

Recent research has established ATF5 as a regulator of exercise-induced mitochondrial quality control:

Mitochondrial content versus function:

• Studies have shown that lack of ATF5 increases mitochondrial content but reduces function

• Use ATF5 antibodies to correlate protein levels with mitochondrial parameters measured by complementary techniques

Exercise-induced adaptations:

• ATF5 is required for normal mitophagic and UPRmt mRNA responses to exercise

• Design protocols that compare sedentary versus exercise conditions with appropriate timing of sample collection

Methodological approaches:

• Combine ATF5 immunoprecipitation with mass spectrometry to identify exercise-induced binding partners

• Develop ChIP-seq protocols using ATF5 antibodies to identify genomic binding sites that respond to exercise

• Employ proximity ligation assays to visualize interactions between ATF5 and mitochondrial proteins

Therapeutic implications:

• Investigate whether exercise-induced improvements in mitochondrial health are ATF5-dependent

• Explore small molecule modulators of ATF5 and test effects on mitochondrial quality control