Recombinant Human MED19 Protein: Handling & Application Protocol

Recombinant Human MED19 Protein is a critical subunit of the Mediator transcriptional coactivator complex, and this protocol provides researchers with authoritative guidance on its proper reconstitution, storage, and use in biochemical and cell-based assays.

Product Name

Recombinant Human MED19 Protein

SKU

rp366225

Protein Function

Subunit of the Mediator complex; involved in transcriptional coactivation and RNA polymerase II regulation

Recommended Storage

Store at -80°C; avoid repeated freeze-thaw cycles

Application Areas

Protein interaction studies, transcription assays, Western blot, ELISA

Host/Expression System

To be confirmed from datasheet; flag for review

Background and Biological Significance of MED19

MED19 (Mediator Complex Subunit 19) is an integral component of the Mediator multiprotein complex, which serves as a master regulator of RNA polymerase II-dependent transcription in eukaryotes. The Mediator complex acts as a molecular bridge between gene-specific transcription factors bound at enhancers and the general transcription machinery assembled at gene promoters.

MED19 is located within the middle module of the Mediator complex and contributes to the structural integrity of the complex as well as its ability to modulate transcriptional activation and repression. Studies have implicated MED19 in the regulation of developmental gene expression programs and have linked its dysregulation to oncogenic processes in several cancer types.

Recombinant Human MED19 Protein produced for research use enables scientists to dissect the molecular mechanisms of Mediator-dependent transcription, identify protein–protein interaction partners, and develop biochemical assays relevant to transcriptional diseases.

Materials and Equipment Required

Before beginning the protocol, ensure the following materials and equipment are available:

• Recombinant Human MED19 Protein (Aladdin Scientific, SKU: rp366225)
• Recommended reconstitution buffer (e.g., sterile phosphate-buffered saline, pH 7.4, with carrier protein such as 0.1% BSA if required — confirm with product datasheet)
• Sterile, low-protein-binding microcentrifuge tubes (1.5 mL)
• Micropipettes and calibrated tips
• Benchtop microcentrifuge
• -80°C freezer for long-term storage; -20°C freezer for short-term storage
• Dry ice or liquid nitrogen for transport and aliquoting
• SDS-PAGE and Western blot apparatus (for quality verification)
• Protein quantification reagents (e.g., BCA or Bradford assay kit)

Note: All specific buffer compositions and concentrations should be verified against the current product datasheet supplied with lot rp366225.

Step-by-Step Reconstitution and Aliquoting Procedure

Follow these steps carefully to maximize protein activity and stability:

1. Thawing: Remove the vial from -80°C storage and allow it to thaw slowly on ice (approximately 10–15 minutes). Do not thaw at room temperature or in a water bath to avoid protein degradation.
2. Brief centrifugation: Centrifuge the vial briefly (1,000–2,000 × g, 30 seconds) to consolidate the lyophilized material or any condensate at the bottom of the tube.
3. Reconstitution: Add the volume of reconstitution buffer specified in the product datasheet dropwise along the wall of the vial. Gently swirl — do not vortex — to ensure complete dissolution. Allow the solution to stand on ice for 5 minutes.
4. Protein quantification: Determine protein concentration using a BCA or Bradford assay against a bovine serum albumin standard curve.
5. Aliquoting: Divide the reconstituted protein into single-use aliquots in low-protein-binding tubes to prevent activity loss from repeated freeze-thaw cycles. Label each aliquot with the date, lot number, and concentration.
6. Storage: Snap-freeze aliquots in liquid nitrogen or on dry ice, then transfer to -80°C for long-term storage. For use within 1–2 weeks, aliquots may be stored at -20°C.

Critical: Avoid more than one freeze-thaw cycle per aliquot. Protein stability data specific to this lot should be confirmed from the datasheet.

Quality Control and Verification

Prior to use in critical experiments, verify the identity and purity of Recombinant Human MED19 Protein using the following methods:

• SDS-PAGE: Resolve a small aliquot under reducing and non-reducing conditions. The band should appear at the expected molecular weight consistent with the MED19 sequence plus any expression tags (confirm expected molecular weight from the datasheet for SKU rp366225).
• Western Blot: Confirm protein identity using an anti-MED19 antibody or an anti-tag antibody appropriate to the expression construct.
• Protein purity assessment: Densitometric analysis of Coomassie-stained SDS-PAGE gels can be used to estimate purity. Target purity should be confirmed from the certificate of analysis (CoA) provided with each lot.
• Functional assay (optional): Where applicable, incorporate MED19 protein into an in vitro Mediator complex assembly assay or a protein–protein interaction assay (e.g., co-immunoprecipitation or pull-down) to confirm biological activity.

Important: Always consult the lot-specific Certificate of Analysis for purity, concentration, and QC results before use.

Experimental Applications and Troubleshooting

Recommended Applications:

• Protein–protein interaction studies: Use Recombinant Human MED19 Protein as bait or prey in pull-down or co-immunoprecipitation experiments to map interactions within the Mediator complex or with transcription factors.
• In vitro transcription assays: Incorporate MED19 into reconstituted transcription systems to study its contribution to Mediator-dependent transcriptional activation.
• Structural studies: Use as a component for cryo-EM or X-ray crystallography studies of Mediator sub-modules.
• Immunogen/standard: Use as a positive control antigen in ELISA or Western blot assay development.

Troubleshooting Guide:

Problem	Likely Cause	Recommended Action
Protein precipitation after reconstitution	Incorrect buffer, too rapid addition, or protein aggregation	Centrifuge at low speed; try alternative buffer with mild detergent; ensure reconstitution on ice
Low or no activity in functional assay	Freeze-thaw damage or incorrect working concentration	Use freshly thawed aliquot; optimize concentration range
Multiple bands on SDS-PAGE	Degradation or incomplete denaturation	Add protease inhibitors; ensure complete denaturation before loading
Weak signal on Western blot	Insufficient protein loading or antibody incompatibility	Increase protein amount; validate antibody specificity for recombinant form

Frequently asked questions

What is the recommended storage condition for Recombinant Human MED19 Protein?

Recombinant Human MED19 Protein should be stored at -80°C for long-term stability. Once reconstituted, divide into single-use aliquots and avoid repeated freeze-thaw cycles, which can significantly reduce protein activity. For short-term use within 1–2 weeks, storage at -20°C is acceptable.

What experimental applications is Recombinant Human MED19 Protein validated for?

Recombinant Human MED19 Protein is suitable for a range of applications including protein–protein interaction studies (pull-down, co-immunoprecipitation), in vitro transcription assays, structural biology experiments (cryo-EM, crystallography), and as an antigen standard in Western blot or ELISA development. Users should confirm validated applications from the lot-specific datasheet provided with SKU rp366225.

How should I reconstitute Recombinant Human MED19 Protein to maintain activity?

Thaw the vial slowly on ice, briefly centrifuge to collect the contents, then add the reconstitution buffer specified in the product datasheet dropwise along the vial wall. Gently swirl — do not vortex — and allow to stand on ice for 5 minutes before use. Always consult the datasheet for the recommended buffer composition and final concentration.

What is the biological role of MED19 in transcription?

MED19 is a subunit of the Mediator complex, a large multiprotein assembly that acts as a transcriptional coactivator for RNA polymerase II in eukaryotes. It resides in the middle module of the Mediator complex and contributes to its structural organization and functional coupling between upstream transcription factors and the basal transcription machinery at gene promoters.

How can I verify the quality of the reconstituted MED19 protein before use?

Quality can be assessed by SDS-PAGE to confirm the expected molecular weight band, followed by Western blot using an anti-MED19 or anti-tag antibody to verify protein identity. Purity estimation by densitometry and protein concentration measurement by BCA or Bradford assay are also recommended. Refer to the Certificate of Analysis (CoA) provided with each lot for lot-specific quality data.

References

1. Conaway RC, Conaway JW. Function and regulation of the Mediator complex. Current Opinion in Genetics & Development. 2011;21(2):225-230. doi:10.1016/j.gde.2011.01.013
2. Allen BL, Taatjes DJ. The Mediator complex: a central integrator of transcription. Nature Reviews Molecular Cell Biology. 2015;16(3):155-166. doi:10.1038/nrm3951
3. Tsai KL, Tomomori-Sato C, Sato S, Conaway RC, Conaway JW, Bhatt DL, Bhatt DL. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex. Cell. 2014;157(6):1430-1444. doi:10.1016/j.cell.2014.05.015

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