Recombinant Human ATRIP Protein: Handling & Usage Protocol

Recombinant Human ATRIP Protein is a critical reagent for investigating the ATR-mediated DNA damage checkpoint pathway, and this protocol ensures its optimal reconstitution, storage, and functional deployment in your assays.

Product Name

Recombinant Human ATRIP Protein

SKU

rp367058

Protein Function

ATRIP (ATR-Interacting Protein) is an obligate binding partner of ATR kinase, essential for ATR recruitment to RPA-coated ssDNA at sites of DNA damage and replication stress.

Recommended Storage

-80°C for long-term storage; avoid repeated freeze-thaw cycles by preparing single-use aliquots.

Reconstitution

Reconstitute in sterile, ultrapure water or an appropriate buffer (e.g., PBS with 0.1% BSA) as recommended in the product datasheet.

Key Research Applications

Biochemical binding assays, ATR-ATRIP complex reconstitution studies, DNA damage checkpoint research, and western blot positive control.

Background: ATRIP and the ATR-Mediated DNA Damage Response

ATRIP (ATR-Interacting Protein) is a key regulatory protein in the DNA damage response (DDR) pathway. It forms a stable heterodimeric complex with ATR (Ataxia Telangiectasia and Rad3-related) kinase, a master regulator of the replication stress response. Upon DNA damage—particularly single-stranded DNA (ssDNA) generated at stalled replication forks or resected double-strand breaks—RPA (Replication Protein A) rapidly coats the exposed ssDNA. ATRIP directly recognizes RPA-ssDNA complexes, thereby recruiting the ATR-ATRIP complex to sites of DNA damage. This recruitment event is a pivotal initiating step for activating downstream checkpoint effectors, including the phosphorylation of CHK1 kinase, which halts cell cycle progression to allow time for DNA repair.

Recombinant Human ATRIP Protein (rp367058) enables researchers to reconstitute these molecular events in vitro, providing a defined and controllable system for studying ATR-ATRIP complex assembly, protein-protein interactions, and checkpoint activation mechanisms.

Materials Required

• Recombinant Human ATRIP Protein, rp367058 (Aladdin Scientific)
• Sterile, nuclease-free ultrapure water (18.2 MΩ·cm)
• Reconstitution buffer: phosphate-buffered saline (PBS, pH 7.4) supplemented with 0.1% BSA (bovine serum albumin) as carrier protein, or as specified in the product datasheet
• Low-protein-binding microcentrifuge tubes (1.5 mL)
• Calibrated micropipettes and sterile tips
• Benchtop microcentrifuge
• Dry ice or liquid nitrogen for snap-freezing aliquots
• -80°C freezer for long-term storage
• BCA or Bradford protein assay kit (for concentration verification)
• SDS-PAGE and western blot reagents (optional, for quality control)

Step-by-Step Protocol: Reconstitution, Aliquoting, and Storage

Step 1 – Pre-Reconstitution Preparation

1. Remove the vial from -80°C storage and allow it to equilibrate briefly on ice (do not thaw at room temperature to minimize protein degradation).
2. Briefly centrifuge the vial (1,000 × g, 10 s) to consolidate any lyophilized material at the bottom.
3. Prepare all reconstitution buffers and low-binding tubes in advance; keep on ice.

Step 2 – Reconstitution

1. Add the recommended volume of sterile ultrapure water or PBS + 0.1% BSA buffer directly to the vial, as specified in the product datasheet. Pipette gently along the vial wall; do not vortex.
2. Allow the vial to stand on ice for 5–10 minutes to permit full dissolution.
3. Gently roll the vial between your palms for 10–15 seconds to ensure homogeneity; inspect visually for particulates.

Step 3 – Concentration Verification (Recommended)

1. Remove a small aliquot (typically 1–2 µL) for protein concentration determination using a BCA or Bradford assay.
2. Compare the measured concentration with the certificate of analysis (CoA) value to confirm expected recovery.

Step 4 – Aliquoting

1. Divide the reconstituted protein into single-use aliquots in low-binding microcentrifuge tubes. Aliquot size should reflect your typical experimental consumption to avoid surplus that must be re-frozen.
2. Snap-freeze aliquots on dry ice or in liquid nitrogen immediately after preparation.

Step 5 – Storage

1. Store snap-frozen aliquots at -80°C. Avoid repeated freeze-thaw cycles, as each cycle can significantly reduce protein activity and integrity.
2. For short-term use (within 24–48 hours), aliquots may be kept at 4°C; prolonged storage at 4°C is not recommended.

Experimental Applications and Integration

Recombinant Human ATRIP Protein (rp367058) is suitable for a range of in vitro research applications:

• Protein-Protein Interaction Studies: Use ATRIP as bait or prey in pull-down or co-immunoprecipitation experiments to map interactions with ATR, RPA70 N-terminal domain, and other DDR factors.
• Biochemical Reconstitution: Reconstitute the ATR-ATRIP heterodimer with recombinant ATR to study kinase activation, substrate phosphorylation (e.g., CHK1 Ser317/Ser345), and regulation by TopBP1 or ETAA1 activators.
• ELISA and Binding Assays: Use as a capture or detection antigen in sandwich ELISA formats to validate antibody specificity or measure binding affinities.
• Western Blot Positive Control: Include a defined quantity of recombinant ATRIP alongside cell lysates to confirm antibody performance and enable band-size verification at the expected molecular weight.
• Structural and Biophysical Studies: Suitable as a component for SPR (surface plasmon resonance), ITC (isothermal titration calorimetry), or cryo-EM sample preparation, subject to purity and buffer compatibility verification.

Note: Confirm specific application suitability against the product datasheet and certificate of analysis prior to experimental use.

Troubleshooting Guide

Problem	Possible Cause	Suggested Solution
Protein does not dissolve completely	Insufficient reconstitution volume or temperature too low	Increase buffer volume slightly; allow additional equilibration time on ice; roll gently—do not vortex
Lower than expected protein concentration	Protein loss due to adsorption to tube walls	Use low-protein-binding tubes; add carrier protein (BSA) to reconstitution buffer
Loss of activity after storage	Freeze-thaw cycling or improper storage temperature	Ensure aliquots are snap-frozen and stored at -80°C; use single-use aliquots
Non-specific bands on western blot	Protein aggregation or degradation	Centrifuge reconstituted protein at 10,000 × g for 5 min at 4°C before use; verify storage conditions
No detectable ATR interaction in pull-down	Incorrect buffer ionic strength or missing cofactors	Optimize salt concentration (typically 100–150 mM NaCl); include ATP and Mg²⁺ if kinase activity is required

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human ATRIP Protein (rp367058)?

The protein should be reconstituted in sterile ultrapure water or PBS (pH 7.4) supplemented with 0.1% BSA as a carrier protein to minimize adsorptive losses. Always refer to the specific product datasheet for the most current reconstitution instructions, as formulation details may be lot-dependent.

How should I store Recombinant Human ATRIP Protein to preserve its activity?

For long-term preservation, store single-use aliquots at -80°C after snap-freezing on dry ice or liquid nitrogen. Avoid repeated freeze-thaw cycles, as these progressively degrade protein integrity and activity. For experiments within 24–48 hours, aliquots may be kept at 4°C, but prolonged refrigerator storage is not advised.

What are the primary research applications for Recombinant Human ATRIP Protein?

Recombinant Human ATRIP Protein is primarily used for studying the ATR-ATRIP checkpoint complex in DNA damage response research. Key applications include in vitro binding assays with ATR and RPA, biochemical reconstitution of the ATR-ATRIP heterodimer, western blot positive controls, ELISA antigen preparation, and biophysical interaction studies such as SPR or ITC.

Why is ATRIP considered essential for ATR kinase activity?

ATRIP is an obligate binding partner of ATR; the two proteins are expressed and function as a stable heterodimeric complex. ATRIP contains an RPA-binding domain that mediates the recruitment of the entire ATR-ATRIP complex to RPA-coated ssDNA at damage sites. Without ATRIP, ATR cannot efficiently localize to sites of DNA damage and fails to activate the checkpoint cascade, including CHK1 phosphorylation.

How can I verify the quality of reconstituted ATRIP protein before use in sensitive assays?

We recommend two quality-control steps: first, determine protein concentration using a BCA or Bradford assay and compare against the CoA-stated value to confirm expected recovery. Second, run a small aliquot on SDS-PAGE under reducing conditions and verify a single predominant band at the expected molecular weight. An optional western blot with a validated anti-ATRIP antibody provides confirmatory identification.

References

1. Cortez D, Guntuku S, Qin J, Elledge SJ. ATR and ATRIP: partners in checkpoint signaling. Science. 2001;294(5547):1713-1716. doi:10.1126/science.1065521
2. Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science. 2003;300(5625):1542-1548. doi:10.1126/science.1083430
3. Ball HL, Cortez D. ATRIP oligomerization is required for ATR-dependent checkpoint signaling. Journal of Biological Chemistry. 2005;280(36):31390-31396. doi:10.1074/jbc.M504961200
4. Aladdin Scientific. Recombinant Human ATRIP Protein, SKU rp367058. Product Datasheet.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.