Recombinant Human CHMP2B Protein: Protocol & Usage Guide

Recombinant Human CHMP2B Protein is a key ESCRT-III pathway component produced for in vitro research applications including binding assays, functional studies of multivesicular body biogenesis, and investigation of neurodegeneration-associated endosomal sorting mechanisms.

Product Name

Recombinant Human CHMP2B Protein

SKU

rp377136

Protein Family

ESCRT-III / Charged Multivesicular Body Protein family

Species Origin

Human (Homo sapiens)

Primary Application

In vitro functional assays, binding studies, ESCRT-III pathway research

Storage Recommendation

Avoid repeated freeze-thaw cycles; store aliquots at -80 °C for long-term use

Background: CHMP2B and the ESCRT-III Pathway

CHMP2B (Charged Multivesicular Body Protein 2B) is a core subunit of the Endosomal Sorting Complexes Required for Transport III (ESCRT-III) machinery. This highly conserved protein plays a central role in membrane remodeling events, including multivesicular body (MVB) biogenesis, cytokinetic abscission, and autophagosome–lysosome fusion. CHMP2B assembles into oligomeric filaments that constrict and sever membrane necks in a topology-independent manner.

Mutations in the human CHMP2B gene have been linked to familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), making CHMP2B a protein of significant interest in neurodegeneration research. Recombinant Human CHMP2B Protein enables in vitro dissection of these molecular events.

Reconstitution Protocol

Follow these steps to reconstitute Recombinant Human CHMP2B Protein (rp377136) for experimental use:

1. Equilibration: Allow the vial to equilibrate to room temperature for 10–15 minutes before opening. Do not heat the vial.
2. Centrifugation: Briefly centrifuge the vial (1,000–2,000 × g, 30 seconds) to pellet any lyophilized material that may have collected on the cap or sides.
3. Reconstitution buffer: Reconstitute the protein in sterile, deionized water or an appropriate buffer (e.g., PBS, pH 7.4, with a carrier protein such as 0.1% BSA if required for stability). Add the reconstitution volume recommended on the Certificate of Analysis (CoA) to achieve the desired working concentration.
4. Gentle mixing: Mix gently by pipetting or slow vortex. Do not vigorously vortex, as this may denature the protein.
5. Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles, which degrade activity.

Note: Exact reconstitution buffer, volume, and concentration should be confirmed against the lot-specific CoA supplied with the product, as these parameters may vary.

Storage and Stability Guidelines

Proper storage is critical to preserving the biological activity of Recombinant Human CHMP2B Protein:

• Short-term storage (≤1 week): Store at 2–8 °C in a refrigerator. Ensure the vial is sealed tightly to prevent contamination and evaporation.
• Long-term storage: Store at –80 °C. Aliquots prepared during reconstitution should be snap-frozen in liquid nitrogen before transfer to –80 °C storage.
• Freeze-thaw cycles: Limit to a maximum of 2–3 cycles. Each freeze-thaw event can reduce protein activity by promoting aggregation or denaturation.
• Carrier protein: Adding a carrier protein (e.g., 0.1–0.5% BSA) to the reconstituted stock may enhance stability during storage, particularly at low protein concentrations.
• Avoid: Storing at room temperature for extended periods, exposure to direct light, or repeated warming and re-freezing.

Refer to the lot-specific CoA for the validated stability data and recommended shelf life after reconstitution.

Recommended Applications and Assay Tips

Recombinant Human CHMP2B Protein is suitable for a range of in vitro research applications:

• Protein–protein interaction studies: CHMP2B interacts with other ESCRT-III subunits (e.g., CHMP4B, VPS4) and with charged lipid membranes. Pull-down assays and co-immunoprecipitation can be used to map these interactions using the recombinant protein as bait or prey.
• Biochemical reconstitution: CHMP2B can be incorporated into minimal ESCRT-III reconstitution systems on giant unilamellar vesicles (GUVs) or supported lipid bilayers to study membrane deformation and scission.
• SDS-PAGE and Western blot validation: Prior to functional assays, confirm protein integrity by SDS-PAGE. Expect a band consistent with the predicted molecular weight of CHMP2B (approximately 24 kDa for the core domain; full-length is ~26 kDa). Use an anti-CHMP2B antibody for Western blot verification.
• ATPase and disassembly assays: When used in conjunction with VPS4 ATPase, CHMP2B-containing filaments can be disassembled in vitro, providing a functional readout for ESCRT-III dynamics.

Assay tip: Because CHMP2B has a tendency to self-polymerize at higher concentrations, keep working concentrations within the range validated on the CoA and perform assays promptly after reconstitution.

Troubleshooting Common Issues

The following table outlines common problems encountered when working with Recombinant Human CHMP2B Protein and suggested remedies:

Problem	Possible Cause	Suggested Action
Protein precipitation after reconstitution	Incompatible buffer, too-rapid reconstitution, or protein aggregation	Try reconstituting in a buffer with slightly higher salt (150–300 mM NaCl) and add 0.1% BSA; reconstitute slowly at 4 °C with gentle rotation.
Low activity in functional assay	Repeated freeze-thaw; degradation during storage	Use freshly reconstituted aliquots; verify protein integrity by SDS-PAGE before assay.
Multiple bands on SDS-PAGE	Protein degradation or contaminants	Check storage conditions; compare with CoA purity data; contact Aladdin Scientific technical support if purity appears below specification.
No signal in Western blot	Antibody incompatibility or incorrect molecular weight expectation	Confirm antibody recognizes recombinant form; check reducing vs. non-reducing conditions; expected MW ~24–26 kDa.

Frequently asked questions

What is the recommended working concentration of Recombinant Human CHMP2B Protein for in vitro binding assays?

The optimal working concentration depends on the specific assay format and should be titrated empirically. As a starting point, concentrations in the range of 0.1–1 µg/mL are commonly used for ELISA-based binding assays, while biochemical reconstitution experiments may require higher amounts. Always refer to the lot-specific CoA for validated concentration ranges.

Can Recombinant Human CHMP2B Protein be used in cell-based assays?

Recombinant Human CHMP2B Protein is primarily intended for in vitro biochemical and biophysical assays. Use in cell-based assays is possible with appropriate delivery methods (e.g., protein transfection reagents or microinjection), but such applications should be validated by the end user. The protein has not been specifically validated for direct cell-based use by Aladdin Scientific.

How many freeze-thaw cycles can Recombinant Human CHMP2B Protein withstand?

It is generally recommended to limit freeze-thaw cycles to no more than 2–3 times to preserve protein activity and prevent aggregation. To minimize freeze-thaw events, reconstituted protein should be divided into small, single-use aliquots and stored at –80 °C.

What is the biological significance of CHMP2B mutations?

Mutations in CHMP2B have been identified as causative factors in familial frontotemporal dementia linked to chromosome 3 (FTD3) and have also been associated with amyotrophic lateral sclerosis (ALS). These mutations disrupt normal ESCRT-III function and impair endosomal sorting and autophagy pathways, leading to neurodegeneration. Recombinant CHMP2B protein enables mechanistic investigation of these disease-associated variants in vitro.

Is a Certificate of Analysis (CoA) available for rp377136?

Yes, a lot-specific Certificate of Analysis is available for Recombinant Human CHMP2B Protein (rp377136). The CoA contains information on purity, concentration, formulation, and functional validation data. Please contact Aladdin Scientific customer service or visit the product page to request the CoA for your specific lot number.

References

1. Skibinski G, et al. Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nature Genetics. 2005;37(8):806–808. doi:10.1038/ng1609
2. Hurley JH, Hanson PI. Membrane budding and scission by the ESCRT machinery: it's all in the neck. Nature Reviews Molecular Cell Biology. 2010;11(8):556–566. doi:10.1038/nrm2937
3. Wollert T, Hurley JH. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature. 2010;464(7290):864–869. doi:10.1038/nature08849
4. Aladdin Scientific. Recombinant Human CHMP2B Protein, SKU rp377136. Product datasheet. Aladdin Scientific; [cited 2024].

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