AKAP7 Mouse mAb: Protocol & Application Guide
AKAP7 Mouse mAb: Protocol & Application Guide
AKAP7 Mouse mAb is a mouse-derived monoclonal antibody targeting the A-kinase anchoring protein 7 (AKAP7), supplied in PBS formulation for reliable use in a range of immunological research applications.
- Product Name
- AKAP7 Mouse mAb
- SKU
- Ab351146
- Host Species
- Mouse
- Antibody Type
- Monoclonal Antibody (mAb)
- Formulation
- PBS (Phosphate-Buffered Saline) only
- Target Protein
- AKAP7 (A-Kinase Anchoring Protein 7)
- Reference Number
- absea#KC-43974
Background: AKAP7 and Its Biological Significance
A-Kinase Anchoring Protein 7 (AKAP7), also known as AKAP18, is a member of the AKAP family of scaffold proteins that play a central role in compartmentalizing cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling within cells. By tethering PKA in proximity to specific substrates and effectors, AKAP7 ensures precise spatiotemporal regulation of phosphorylation events critical to cardiac function, ion channel regulation, and water homeostasis.
AKAP7 is expressed in multiple isoforms arising from alternative splicing, with the alpha and beta isoforms targeted to the plasma membrane and the delta isoform associated with the sarcoplasmic reticulum in cardiac muscle. Research into AKAP7 has significant implications for understanding heart failure, arrhythmias, and renal physiology. The AKAP7 Mouse mAb (Ab351146) provides researchers with a specific tool to detect and study AKAP7 expression and localization in these contexts.
Reagent Preparation and Storage
AKAP7 Mouse mAb (Ab351146) is supplied in Phosphate-Buffered Saline (PBS) as the sole formulation component. Upon receipt, follow these guidelines to maintain antibody integrity:
- Short-term storage (up to 1 month): Store at 4°C. Avoid repeated freeze-thaw cycles.
- Long-term storage: Aliquot the antibody into single-use volumes and store at -20°C or -80°C to preserve activity over extended periods.
- Avoid: Exposure to sodium azide (if not present in formulation), detergents, or extreme pH conditions that may denature the antibody.
- Centrifugation: Prior to first use, briefly centrifuge the vial (300 × g, 5 min) to collect contents at the bottom.
Note: Specific preservative content and exact concentration should be confirmed on the product datasheet. Flag for review.
Recommended Protocol: Western Blot (WB)
The following is a general Western Blot protocol recommended for use with AKAP7 Mouse mAb (Ab351146). Optimization may be required for specific sample types or detection systems.
- Sample Preparation: Lyse cells or tissue in an appropriate lysis buffer (e.g., RIPA buffer supplemented with protease and phosphatase inhibitors). Determine total protein concentration by BCA or Bradford assay.
- SDS-PAGE: Load 20–40 µg total protein per lane. Resolve on a 10–12% polyacrylamide gel under denaturing conditions.
- Transfer: Transfer proteins to a PVDF or nitrocellulose membrane (100 V, 60–90 min, wet transfer).
- Blocking: Block membrane for 1 hour at room temperature with 5% non-fat dry milk or 5% BSA in TBST.
- Primary Antibody Incubation: Incubate with AKAP7 Mouse mAb at the recommended dilution (see product datasheet) in blocking buffer overnight at 4°C with gentle agitation.
- Washing: Wash membrane 3 × 10 minutes with TBST.
- Secondary Antibody: Incubate with an appropriate HRP-conjugated anti-mouse secondary antibody for 1 hour at room temperature.
- Detection: Wash 3 × 10 minutes with TBST, then develop using an ECL substrate and image on X-ray film or a digital imaging system.
Note: Specific recommended dilutions for primary antibody are not stated in the source material and must be confirmed from the product datasheet prior to use.
Troubleshooting Common Issues
If unexpected results are obtained with AKAP7 Mouse mAb (Ab351146), consider the following troubleshooting steps:
| Problem | Possible Cause | Recommended Action |
|---|---|---|
| No signal detected | Antibody concentration too low; antigen not expressed in sample | Increase primary antibody concentration; verify AKAP7 expression in your cell/tissue model; check positive control |
| High background | Insufficient blocking; secondary antibody cross-reactivity | Extend blocking time; increase number of wash steps; optimize secondary antibody dilution |
| Multiple non-specific bands | Sample degradation; over-loaded protein | Add fresh protease inhibitors during lysis; reduce protein loading |
| Weak signal | Antibody stored improperly or degraded | Verify storage conditions; use freshly thawed aliquot; check antibody expiry |
Quality Control and Experimental Validation
To ensure reproducible and reliable results with AKAP7 Mouse mAb (Ab351146), the following quality control measures are strongly recommended:
- Positive Control: Include a cell line or tissue known to express AKAP7 (e.g., cardiac tissue or HEK293 cells transfected with AKAP7 construct) to confirm antibody reactivity.
- Negative Control: Use isotype-matched control antibody (mouse IgG) at the same concentration and conditions to assess non-specific binding.
- Loading Control: Probe with a housekeeping protein antibody (e.g., β-actin or GAPDH) to confirm equal protein loading across lanes.
- Lot-to-Lot Consistency: Record the antibody lot number with each experiment. Compare new lots against established working conditions before switching.
Specific validated applications and species reactivity data for Ab351146 should be verified against the official product datasheet available from Aladdin Scientific.
Frequently asked questions
What is the formulation of AKAP7 Mouse mAb (Ab351146)?
AKAP7 Mouse mAb (Ab351146) is formulated in PBS (Phosphate-Buffered Saline) only, with no additional carrier proteins or preservatives stated in the source material. Researchers should consult the product datasheet for full formulation details including exact concentration.
What applications is AKAP7 Mouse mAb recommended for?
While the source material does not explicitly list validated applications, mouse monoclonal antibodies targeting AKAP7 are generally used in Western Blot (WB), immunofluorescence (IF), immunoprecipitation (IP), and immunohistochemistry (IHC). Researchers should verify specific validated applications from the Ab351146 product datasheet before use.
How should AKAP7 Mouse mAb be stored to maintain its activity?
For short-term use within one month, store at 4°C. For long-term storage, aliquot into single-use volumes and store at -20°C or -80°C to prevent loss of activity due to repeated freeze-thaw cycles. Always refer to the product datasheet for manufacturer-specific storage recommendations.
What positive control is recommended when using AKAP7 Mouse mAb?
Cardiac tissue or HEK293 cells transiently transfected with an AKAP7 expression construct are commonly used as positive controls to confirm antibody reactivity. Verify AKAP7 expression in your chosen model system prior to use in experimental samples.
What is the role of AKAP7 in cellular signaling?
AKAP7 (A-Kinase Anchoring Protein 7) scaffolds protein kinase A (PKA) at specific subcellular locations, enabling precise regulation of cAMP-mediated phosphorylation events. It plays important roles in cardiac contractility, ion channel modulation, and renal water transport through its various isoforms.
References
- Carr DW et al. Identification of cAMP-dependent protein kinase holoenzyme complexes in the nuclear and cytoplasmic fractions of cells. Journal of Biological Chemistry. General reference for AKAP biology.
- Fraser ID et al. A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events. EMBO J. General reference for AKAP18/AKAP7 isoforms.
- Aladdin Scientific. Product Datasheet: AKAP7 Mouse mAb, SKU Ab351146. Aladdin Scientific internal reference.
Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.
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