Recombinant Human GAS2L2 Protein: Protocol & Usage Guide

Recombinant Human GAS2L2 Protein is a research-grade cytoskeletal regulatory protein produced for use in cell biology assays, structural studies, and investigations of microtubule and actin filament dynamics.

Product Name

Recombinant Human GAS2L2 Protein

SKU

rp372867

Species

Human (Homo sapiens)

Protein Family

GAS2-like protein family; contains CH (calponin homology) and GAR (GAS2-related) domains

Primary Research Application

Cytoskeletal dynamics, microtubule/actin binding assays, cell cycle studies

Recommended Storage

-80 °C for long-term storage; avoid repeated freeze-thaw cycles

Format

Recombinant protein supplied lyophilized or in solution (confirm with datasheet)

Background & Biological Significance of GAS2L2

GAS2L2 (Growth Arrest-Specific Protein 2-Like 2) is a member of the GAS2-like protein family, which plays critical roles in coordinating cytoskeletal organization and cell cycle progression. The protein contains two conserved structural domains: a calponin homology (CH) domain responsible for actin filament binding, and a GAS2-related (GAR) domain that mediates interactions with microtubules. Through these dual binding capacities, GAS2L2 serves as a molecular bridge linking the actin and microtubule networks.

GAS2L2 has been implicated in the regulation of the mitotic spindle, centrosome function, and ciliogenesis. Research suggests that GAS2L2 participates in controlling cell division fidelity, and dysregulation of GAS2-like proteins has been associated with genomic instability. Recombinant Human GAS2L2 Protein enables researchers to dissect these molecular mechanisms in vitro using defined, high-purity protein preparations.

Reagent Preparation & Reconstitution

Materials Required:

• Recombinant Human GAS2L2 Protein (rp372867)
• Sterile deionized water or manufacturer-recommended reconstitution buffer
• Low-protein-binding microcentrifuge tubes
• Ice bucket
• Calibrated micropipettes

Reconstitution Procedure:

1. Allow the lyophilized protein vial to equilibrate to room temperature for 10–15 minutes before opening to prevent moisture condensation. If supplied in solution, thaw gently on ice.
2. Briefly centrifuge the vial (1,000 × g, 30 seconds) to collect any powder at the bottom.
3. Add the appropriate volume of sterile reconstitution buffer (typically PBS pH 7.4 or the buffer specified in the product datasheet) along the wall of the vial. Do not pipette directly onto the lyophilized cake.
4. Gently swirl or invert the vial to dissolve. Do not vortex, as this may denature the protein.
5. Allow the solution to stand on ice for 5 minutes to ensure complete dissolution.
6. Aliquot the reconstituted protein into single-use volumes using low-protein-binding tubes to minimize freeze-thaw degradation. Store aliquots at -80 °C.

Note: Always consult the product-specific datasheet (absea#PP-6833) for lot-specific reconstitution volumes and buffer recommendations.

Experimental Protocol: Cytoskeletal Binding Assays

Recombinant Human GAS2L2 Protein is particularly suited for in vitro co-sedimentation assays to study its interaction with F-actin and microtubules. The following generalized workflow outlines a co-sedimentation approach:

F-Actin Co-Sedimentation Assay (Outline):

1. Polymerize actin monomers (G-actin) into filamentous actin (F-actin) in polymerization buffer (e.g., 10 mM Tris-HCl pH 7.5, 2 mM MgCl₂, 1 mM ATP, 50 mM KCl) for 1 hour at room temperature.
2. Mix reconstituted GAS2L2 protein with preformed F-actin at the desired molar ratios. Incubate for 30 minutes at room temperature.
3. Ultracentrifuge the reaction mixture (100,000 × g, 30 minutes, 4 °C) to pellet F-actin and any associated proteins.
4. Collect supernatant and resuspend pellet in equal volumes of SDS sample buffer. Analyze both fractions by SDS-PAGE and Coomassie staining or western blot using an anti-GAS2L2 antibody.
5. GAS2L2 detected in the pellet fraction indicates direct actin filament binding.

Microtubule Co-Sedimentation Assay (Outline):

1. Polymerize tubulin in BRB80 buffer (80 mM PIPES pH 6.8, 1 mM MgCl₂, 1 mM EGTA) supplemented with GTP and Taxol to stabilize microtubules.
2. Mix GAS2L2 protein with preformed microtubules and incubate at 37 °C for 20 minutes.
3. Pellet microtubules by centrifugation (100,000 × g, 30 minutes, 37 °C). Analyze supernatant and pellet fractions by SDS-PAGE.

Optimize protein concentrations and incubation conditions empirically. Include appropriate negative controls (protein alone without cytoskeletal polymer).

Storage, Handling & Quality Control

Storage Conditions:

• Store lyophilized protein at -20 °C or -80 °C as specified in the product datasheet.
• Once reconstituted, aliquot immediately and store at -80 °C. Avoid repeated freeze-thaw cycles, which degrade protein activity and integrity.
• Working aliquots may be kept at 4 °C for up to 1 week; verify activity before extended use.

Handling Precautions:

• Always work on ice when handling reconstituted protein to minimize degradation.
• Use low-protein-binding plasticware (tubes and pipette tips) to reduce adsorptive losses.
• Add protease inhibitors to buffers if the protein will be used in complex biological matrices.

Quality Control Recommendations:

• Purity Assessment: Run an aliquot on SDS-PAGE under reducing and non-reducing conditions and stain with Coomassie Blue. Compare the observed molecular weight band to the theoretical molecular weight of GAS2L2 (~75 kDa; confirm with datasheet).
• Identity Confirmation: Western blot using a validated anti-GAS2L2 antibody confirms protein identity.
• Activity Verification: Perform a pilot co-sedimentation experiment with F-actin or microtubules before committing to large-scale studies.
• Endotoxin: If the protein is to be used in cell-based assays, request or verify endotoxin levels (LAL assay result) from the product datasheet.

Troubleshooting

The following table summarizes common issues encountered when working with Recombinant Human GAS2L2 Protein and suggested remedies:

Problem	Possible Cause	Suggested Solution
Protein precipitates upon reconstitution	Incorrect buffer, rapid addition of buffer, or protein aggregation	Use recommended buffer; add buffer slowly along vial wall; gently swirl rather than vortex; centrifuge briefly to remove aggregates
No band or faint band on SDS-PAGE	Insufficient protein loaded; degradation; adsorption to plasticware	Increase loaded amount; check storage history; use low-protein-binding tubes; add BSA carrier if appropriate
No co-sedimentation with F-actin or microtubules	Protein denatured; incorrect assay conditions; suboptimal molar ratios	Verify protein integrity by SDS-PAGE; optimize buffer pH and salt concentration; titrate protein concentrations
High background in western blot	Non-specific antibody binding; insufficient blocking	Optimize blocking (5% non-fat milk or BSA); titrate primary antibody concentration; increase wash stringency

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GAS2L2 Protein?

The reconstitution buffer is specified on the product datasheet for lot rp372867 (absea#PP-6833). As a general guideline, sterile PBS (pH 7.4) supplemented with a carrier protein such as 0.1% BSA is commonly used for recombinant cytoskeletal proteins. Always follow the lot-specific instructions provided with the product.

How many freeze-thaw cycles can Recombinant Human GAS2L2 Protein tolerate?

To preserve protein integrity and activity, repeated freeze-thaw cycles should be avoided. It is strongly recommended to aliquot the reconstituted protein into single-use volumes immediately after reconstitution and store them at -80 °C. This minimizes degradation and ensures reproducible results across experiments.

What assays is Recombinant Human GAS2L2 Protein suitable for?

Recombinant Human GAS2L2 Protein is suitable for in vitro cytoskeletal binding assays including F-actin and microtubule co-sedimentation assays, protein-protein interaction studies, structural biology applications, and as a standard or positive control in western blotting or ELISA. Its dual CH and GAR domains make it a valuable tool for dissecting cytoskeletal regulatory mechanisms.

What is the theoretical molecular weight of GAS2L2 and how should I validate the protein on SDS-PAGE?

The theoretical molecular weight of human GAS2L2 should be confirmed against the product datasheet, as recombinant constructs may include tags or truncations that alter the apparent molecular weight. Run the protein under reducing SDS-PAGE conditions alongside a broad-range molecular weight marker and compare the dominant band to the expected size. Identity can be further confirmed by western blot with a validated anti-GAS2L2 antibody.

Can Recombinant Human GAS2L2 Protein be used in cell-based assays?

Recombinant Human GAS2L2 Protein may be used in cell-based applications, but endotoxin levels must be verified before use to avoid confounding inflammatory responses. Consult the product datasheet for endotoxin testing data. Additionally, a suitable delivery method (e.g., microinjection or protein transfection reagent) may be required for intracellular applications, as GAS2L2 is primarily an intracellular protein.

References

1. Stroud MJ, et al. GAS2-like proteins mediate coordination of the actin and microtubule cytoskeletons. Journal of Cell Science. (General reference for GAS2-like protein family function — specific details require verification.)
2. Aladdin Scientific. Recombinant Human GAS2L2 Protein, SKU rp372867, Product Datasheet absea#PP-6833. Aladdin Scientific.

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