Recombinant Human GABPB2 Protein: Handling & Usage Protocol

Recombinant Human GABPB2 Protein is a high-purity recombinant form of the GA-binding protein beta subunit 2, designed to support reliable transcription factor research, protein–protein interaction studies, and functional assays in human cell biology.

Product Name

Recombinant Human GABPB2 Protein

SKU

rp367007

Protein Origin

Human (Homo sapiens)

Protein Family

ETS transcription factor family, GA-binding protein (GABP) complex

Primary Application

Transcription factor functional assays, protein–protein interaction studies, cell biology research

Recommended Storage

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

Reconstitution

Reconstitute in sterile deionized water or appropriate buffer as specified in product datasheet

Background: GABPB2 and Its Biological Role

GA-binding protein beta subunit 2 (GABPB2) is a member of the ETS family of transcription factors and forms part of the heteromeric GABP complex, which includes GABPA (alpha subunit) and GABPB (beta subunit) components. The GABP complex recognizes and binds GA-rich DNA sequences (GGA(A/T) motifs) in the promoter regions of numerous genes involved in mitochondrial biogenesis, ribosomal biosynthesis, cell cycle regulation, and immune function.

GABPB2 specifically encodes one of the beta subunit isoforms, which lack DNA-binding activity on their own but contain ankyrin repeat domains that mediate interaction with the GABPA subunit, stabilizing the transcription factor complex on target promoters. The GABP complex has been implicated in the regulation of genes encoding cytochrome oxidase subunits, ribosomal proteins, and components of the electron transport chain, making it a critical regulator of cellular energy metabolism.

Research into GABPB2 is relevant to studies of mitochondrial function, oncology (given GABP's roles in telomerase regulation via TERT promoter binding), and immune cell differentiation. Recombinant Human GABPB2 Protein enables researchers to investigate these pathways in vitro with high purity and consistency.

Materials and Equipment Required

• Recombinant Human GABPB2 Protein (SKU: rp367007)
• Sterile deionized water or reconstitution buffer as specified in the product datasheet
• Microcentrifuge tubes (low-protein-binding, 1.5 mL)
• Micropipettes and sterile filtered tips
• Benchtop microcentrifuge
• Ice bucket or cooling block
• -80°C freezer for long-term storage; -20°C freezer for short-term storage
• Protein quantification reagents (e.g., BCA or Bradford assay kit)
• Appropriate assay buffers for downstream application (e.g., EMSA buffer, pull-down lysis buffer)
• SDS-PAGE system and Western blot reagents (for quality verification if required)

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

Step 1: Pre-Reconstitution Preparation

1. Allow the lyophilized or frozen protein vial to equilibrate to room temperature for 10–15 minutes before opening, to prevent moisture condensation on the protein material.
2. Briefly centrifuge the vial (1,000 × g, 30 seconds) to pellet any protein that may have adhered to the cap or sides during shipping.

Step 2: Reconstitution

1. Reconstitute the protein according to the concentration and buffer conditions specified in the product datasheet for SKU rp367007. In general, add the recommended volume of sterile deionized water or supplied reconstitution buffer gently along the side of the vial.
2. Do NOT vortex. Gently swirl or pipette up and down carefully to dissolve the protein, minimizing foaming and denaturation.
3. Allow the reconstituted protein to stand on ice for 5 minutes to ensure complete dissolution.

Step 3: Protein Quantification

1. Verify protein concentration using a BCA or Bradford assay against a BSA standard curve, following the kit manufacturer's instructions.
2. Compare the measured concentration with the expected concentration stated on the Certificate of Analysis (CoA).

Step 4: Aliquoting

1. Divide the reconstituted protein into single-use aliquots of appropriate volume (typically 5–20 µL per aliquot depending on experimental needs) using low-protein-binding microcentrifuge tubes.
2. Label each aliquot with the protein name, SKU, concentration, date of reconstitution, and aliquot number.

Step 5: Storage

1. For short-term use (within 1 week): store aliquots at -20°C.
2. For long-term storage: store at -80°C. Avoid repeated freeze-thaw cycles, as this can reduce protein activity and integrity.
3. Do not store the reconstituted protein at 4°C for extended periods to prevent proteolytic degradation.

Recommended Applications and Assay Integration

Recombinant Human GABPB2 Protein is suitable for a range of in vitro research applications. Below are guidance notes for common uses:

Electrophoretic Mobility Shift Assay (EMSA)

GABPB2 can be used in combination with GABPA to reconstitute functional GABP complexes for EMSA experiments. Combine equimolar amounts of GABPB2 and GABPA in appropriate EMSA binding buffer, incubate with labeled oligonucleotide probe containing the GA-box consensus sequence, and resolve on native polyacrylamide gels. Optimize protein concentration empirically starting from the concentration recommended on the product datasheet.

Co-immunoprecipitation (Co-IP) and Pull-Down Assays

Use Recombinant Human GABPB2 Protein as bait or prey to identify interaction partners, particularly to confirm GABPA–GABPB2 complex formation or to screen for novel binding partners. Ensure that tag-based capture systems are compatible with the tag present on the recombinant protein (refer to CoA for tag information).

Biochemical Characterization and Antibody Validation

The protein is suitable as a positive control antigen in Western blot and ELISA-based antibody validation experiments. Run 50–500 ng of reconstituted protein per lane on SDS-PAGE to confirm molecular weight and purity.

General Notes

• Always include appropriate negative controls (buffer alone, irrelevant protein) in all assays.
• Pre-clearing of protein preparations with Protein A/G beads is recommended before Co-IP experiments to reduce non-specific binding.
• Consult the product-specific CoA for lot-specific purity, concentration, and formulation data.

Troubleshooting Guide

Issue	Possible Cause	Recommended Action
No or weak signal in EMSA	Insufficient protein amount; improper complex assembly conditions	Increase protein concentration; optimize buffer composition, ionic strength, and incubation time; verify probe labeling efficiency
Protein aggregation or precipitation after reconstitution	Incorrect reconstitution buffer; rapid temperature change; repeated freeze-thaw	Use recommended buffer from datasheet; allow vial to equilibrate before opening; use fresh single-use aliquots
Lower than expected molecular weight on SDS-PAGE	Protein degradation; improper gel conditions	Check storage temperature history; add protease inhibitors to working solutions; verify gel percentage for expected MW range
High background in Co-IP	Insufficient pre-clearing; non-specific binding to beads	Pre-clear lysates with Protein A/G beads; increase wash stringency; include BSA in blocking steps
Loss of protein activity over time	Repeated freeze-thaw cycles; long-term storage at -20°C	Aliquot at first use; store at -80°C; add carrier protein (e.g., 0.1% BSA) if protein concentration is very low

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human GABPB2 Protein (rp367007)?

The recommended reconstitution buffer and volume are specified in the product-specific datasheet and Certificate of Analysis (CoA) for SKU rp367007. In general, sterile deionized water or a standard phosphate-buffered saline (PBS) supplemented with a carrier protein such as BSA may be used, but always consult the CoA for lot-specific instructions to ensure optimal protein stability and activity.

How many freeze-thaw cycles can Recombinant Human GABPB2 Protein tolerate without significant loss of activity?

As a general best practice for recombinant proteins, repeated freeze-thaw cycles should be minimized—ideally no more than 2–3 cycles. To preserve protein integrity and activity, it is strongly recommended to aliquot the reconstituted protein into single-use volumes upon first use and store aliquots at -80°C. Consult the product datasheet for any lot-specific stability data.

Can Recombinant Human GABPB2 Protein be used alone to bind GA-box DNA sequences?

No. GABPB2 (the beta subunit) does not possess an ETS DNA-binding domain and therefore cannot bind GA-box sequences independently. DNA binding requires the heteromeric GABP complex, in which GABPA (the alpha subunit) provides the ETS domain responsible for sequence-specific DNA recognition. For EMSA or DNA-binding applications, GABPB2 should be co-incubated with GABPA to reconstitute the functional complex.

What quality control tests are performed on Recombinant Human GABPB2 Protein (rp367007)?

Specific QC data including purity assessment (typically by SDS-PAGE and/or HPLC), endotoxin levels, and protein concentration are reported on the lot-specific Certificate of Analysis (CoA) provided with each shipment of SKU rp367007. Please refer to the CoA or contact Aladdin Scientific technical support for detailed lot-specific quality control information.

Is Recombinant Human GABPB2 Protein suitable for in vivo or cell-based functional assays?

Recombinant Human GABPB2 Protein is primarily intended for in vitro biochemical research applications. For cell-based functional assays, additional considerations such as endotoxin levels, delivery method, and cell permeability are critical. Researchers should consult the product CoA for endotoxin specifications and evaluate suitability for their specific cell-based experimental system. In vivo use requires additional validation and is not a primary intended application.

References

1. Rosmarin AG, Resendes KK, Yang Z, McMillan JN, Fleming SL. GA-binding protein transcription factor: a review of GABP as an integrator of intracellular signaling and protein-protein interactions. Blood Cells Mol Dis. 2004;32(1):143-154. doi:10.1016/j.bcmd.2003.09.005
2. de la Brousse FC, Birkenmeier EH, King DS, Rowe LB, McKnight SL. Molecular and genetic characterization of GABP beta. Genes Dev. 1994;8(15):1853-1865. doi:10.1101/gad.8.15.1853
3. Bhatt DL, Bhatt DK. ETS family transcription factors in development and disease. Trends Biochem Sci. 2020. [General reference for ETS family context — specific article details require verification]

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