Recombinant Human ING1 Protein: Handling & Application Protocol
Recombinant Human ING1 Protein: Handling & Application Protocol
Recombinant Human ING1 Protein is a prokaryotic or eukaryotic expression-derived tumor suppressor protein that plays a critical role in chromatin remodeling, DNA damage response, and apoptosis regulation, making it an essential tool for cancer biology and epigenetics research.
- Product Name
- Recombinant Human ING1 Protein
- SKU
- rp365449
- Gene Name
- ING1 (Inhibitor of Growth Family Member 1)
- Protein Function
- Tumor suppressor; involved in chromatin remodeling, DNA damage response, and apoptosis
- Key Domain
- Plant Homeodomain (PHD) finger — binds trimethylated histone H3K4
- Recommended Storage
- -80°C for long-term storage; avoid repeated freeze-thaw cycles
- Primary Applications
- Chromatin pull-down, ELISA, SDS-PAGE quality control, apoptosis assays, protein interaction studies
Background and Biological Significance of ING1
ING1 (Inhibitor of Growth Family Member 1) is a founding member of the ING family of tumor suppressor proteins. It is encoded by the ING1 gene located on human chromosome 13q34 and is ubiquitously expressed across human tissues. ING1 functions as a critical regulator of cell cycle progression, DNA damage signaling, and programmed cell death.
Structurally, ING1 contains a conserved Plant Homeodomain (PHD) finger at its C-terminus, which selectively recognizes and binds trimethylated lysine 4 on histone H3 (H3K4me3), a mark associated with actively transcribed gene promoters. Through this interaction, ING1 recruits histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to chromatin, thereby regulating target gene expression in a context-dependent manner.
ING1 also interacts with p53 to amplify p53-dependent transcriptional responses following DNA damage, and loss of ING1 expression has been documented in multiple human cancers including breast cancer, melanoma, and gastric cancer. Recombinant Human ING1 Protein enables in vitro biochemical characterization of these functions and serves as a reference standard for antibody validation and interaction studies.
Reconstitution and Storage Guidelines
Reconstitution: Allow the lyophilized protein vial to equilibrate to room temperature for 10–15 minutes before opening. Reconstitute the protein in the volume of sterile, deionized water or the recommended reconstitution buffer indicated on the product datasheet to yield the desired working concentration. Gently pipette to mix; do not vortex, as vigorous agitation can disrupt protein tertiary structure.
Carrier Protein: If the recombinant protein is provided without a carrier, consider adding bovine serum albumin (BSA) to a final concentration of 0.1% to improve stability and prevent adsorption to tube surfaces during dilution and storage.
Aliquoting: Immediately prepare single-use aliquots after reconstitution to avoid repeated freeze-thaw cycles, which can lead to protein aggregation, loss of activity, and reduced assay reproducibility.
Storage Conditions:
- Short-term (up to 1 week): Store at 4°C.
- Long-term: Store at −80°C in a frost-free freezer. Avoid storage at −20°C with frequent door opening cycles.
- Do not expose to repeated freeze-thaw cycles. Recommend no more than 2–3 freeze-thaw cycles maximum.
Shelf Life: Refer to the certificate of analysis (CoA) for lot-specific expiry information. Typically 12 months from date of receipt when stored as recommended.
Step-by-Step Application Protocol
Recombinant Human ING1 Protein is suitable for multiple downstream applications. Below is a generalized workflow applicable to protein interaction and chromatin binding studies.
Materials Required:
- Recombinant Human ING1 Protein (rp365449)
- Binding buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20, 1 mM DTT
- Protease inhibitor cocktail
- BSA (for blocking)
- SDS-PAGE reagents (for quality verification)
Step 1 — Quality Verification by SDS-PAGE: Before use, resolve 1–2 µg of reconstituted protein on a 12% SDS-PAGE gel under reducing conditions. Stain with Coomassie Brilliant Blue and confirm that the observed molecular weight band corresponds to the expected size for ING1 (refer to the product datasheet for the specific isoform molecular weight). A single predominant band indicates high purity.
Step 2 — Protein Interaction / Pull-Down Assay:
- Pre-clear bead-immobilized ING1 protein (or GST/His-tagged bait) in binding buffer containing 1% BSA for 30 minutes at 4°C to reduce nonspecific binding.
- Add prey protein (e.g., p53 or histone peptide) at the molar ratio indicated in your experimental design.
- Incubate at 4°C for 1–2 hours with end-over-end rotation.
- Wash beads 3× with binding buffer (400 µL each) to remove unbound protein.
- Elute bound proteins in SDS loading buffer, boil at 95°C for 5 minutes, and analyze by SDS-PAGE followed by western blot or silver stain.
Step 3 — ELISA-Based Detection: ING1 protein can be used as a coating antigen (0.5–2 µg/mL in carbonate buffer, pH 9.6) for ELISA to screen antibody specificity or to validate custom antibodies. Follow standard indirect ELISA protocols with appropriate blocking (1–5% BSA or non-fat milk) and secondary antibody detection.
Step 4 — Apoptosis-Related Functional Assays: For assays examining ING1-mediated apoptosis, add recombinant ING1 to cell-free apoptosis reconstitution systems or use it as a control standard in immunoprecipitation experiments. Optimize protein concentration empirically for each system.
Troubleshooting and Quality Control
The following guidance addresses common issues encountered when working with Recombinant Human ING1 Protein:
| Issue | Possible Cause | Recommended Action |
|---|---|---|
| Protein aggregation after reconstitution | Rapid dissolution or incorrect buffer | Reconstitute slowly at recommended temperature; centrifuge at 10,000×g for 5 min to pellet aggregates before use |
| Low or absent band on SDS-PAGE | Insufficient protein loaded or incomplete reconstitution | Increase load to 2–5 µg; ensure full dissolution before electrophoresis |
| Multiple bands observed | Protein degradation or contamination | Verify cold-chain integrity; use fresh protease inhibitors; check lot CoA for purity specification |
| Weak signal in pull-down / ELISA | Suboptimal protein concentration or buffer incompatibility | Titrate protein concentration; verify buffer pH and salt concentration; include positive control |
| Loss of activity over time | Repeated freeze-thaw or improper storage | Use single-use aliquots; store at −80°C; minimize time at room temperature |
Always run a positive control (e.g., a well-characterized recombinant protein of known activity) alongside ING1 to validate assay performance. Retain CoA documentation for every lot used in publication-quality experiments.
Safety, Regulatory, and Ordering Information
Safety: Recombinant Human ING1 Protein is intended for in vitro research use only. It is not approved for diagnostic or therapeutic use in humans or animals. Handle with standard laboratory precautions: wear appropriate personal protective equipment (PPE) including gloves and eye protection. Consult the Safety Data Sheet (SDS) provided with the product before use.
Biological Safety: The protein is produced under controlled conditions. However, as with all biological reagents, treat as potentially biohazardous. Dispose of according to your institution's biosafety guidelines and local regulations.
Regulatory Status: For research use only (RUO). Not for use in clinical diagnostics.
Ordering:
- Product: Recombinant Human ING1 Protein
- SKU: rp365449
- Supplier Reference: absea#PP-1020
- Supplier: Aladdin Scientific
Contact your Aladdin Scientific regional representative or visit the Aladdin website to request a quote, check current stock availability, and download the most recent product datasheet and CoA for lot-specific quality parameters including purity (%), endotoxin level, and biological activity data.
Frequently asked questions
What is the recommended reconstitution buffer for Recombinant Human ING1 Protein?
The recommended reconstitution buffer is provided on the product-specific datasheet and certificate of analysis (CoA) supplied with each lot. In general, sterile deionized water or a mild physiological buffer (e.g., PBS pH 7.4 or 50 mM Tris-HCl pH 7.5) is appropriate. Always consult the lot-specific CoA for the exact recommendation, as formulations may vary by production lot.
How many freeze-thaw cycles can Recombinant Human ING1 Protein withstand?
To preserve protein integrity and biological activity, it is strongly recommended to limit freeze-thaw cycles to a maximum of 2–3 times. Repeated freeze-thaw cycles can cause protein aggregation, structural denaturation, and significant loss of functional activity. Prepare single-use aliquots immediately after reconstitution and store at −80°C to avoid unnecessary freeze-thaw events.
What downstream applications is Recombinant Human ING1 Protein validated for?
Recombinant Human ING1 Protein (rp365449) is suitable for applications including SDS-PAGE purity analysis, ELISA (as coating antigen or standard), protein–protein interaction studies (pull-down assays), chromatin binding assays exploiting the PHD finger domain, and as a reference control in western blotting and immunoprecipitation. Specific validated applications and activity data are detailed in the lot-specific CoA and product datasheet available from Aladdin Scientific.
Why is ING1 considered a tumor suppressor, and how does this inform experimental design?
ING1 is classified as a tumor suppressor because its expression is frequently downregulated or lost in multiple human cancers, and it functionally promotes cell cycle arrest and apoptosis while cooperating with p53 in DNA damage responses. When designing experiments with Recombinant Human ING1 Protein, researchers should include appropriate p53-competent or p53-null cell-free systems as controls, and consider the specific ING1 isoform being studied, as different splice variants may exhibit distinct interaction profiles and activities.
How should I verify the quality of Recombinant Human ING1 Protein upon receipt?
Upon receipt, inspect the vial for physical integrity and confirm that the product has been maintained in the cold chain (on dry ice for frozen shipments). After reconstitution, run 1–2 µg on a reducing SDS-PAGE gel and stain with Coomassie Brilliant Blue to confirm the presence of a band at the expected molecular weight with high purity. Cross-reference the result with the purity and molecular weight specifications stated in the lot CoA provided by Aladdin Scientific. Report any discrepancies to technical support promptly.
References
- Garkavtsev I, et al. The candidate tumour suppressor p33ING1 cooperates with p53 in cell growth control. Nature. 1998;391(6664):295-298. doi:10.1038/34675
- Shi X, et al. ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature. 2006;442(7098):96-99. doi:10.1038/nature04835
- Doyon Y, et al. ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation. Molecular Cell. 2006;21(1):51-64. doi:10.1016/j.molcel.2005.12.007
- Aladdin Scientific. Recombinant Human ING1 Protein, SKU rp365449. Product Datasheet. Aladdin Scientific.
Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.