[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[Applications](https://www.sigmaaldrich.com/US/en/applications)[Genomics](https://www.sigmaaldrich.com/US/en/applications/genomics)Cloning & Expression # Cloning & Expression  __Recombinant Protein Expression in Mammalian Cells__ Cloning and gene expression technologies are used by researchers in diverse fields to investigate a wide range of biological questions, including understanding gene function, molecular pathway analysis, embryonic development, disease research, and bioprocessing of biologics and therapeutics to name a few. Once the gene or genetic sequence is identified, researchers must choose the best molecular cloning strategy and cell-based protein expression system based on the specific application need. For additional information on gene expression using CRISPR technology, or gene silencing using RNAi reagents, please visit our [gene expression and gene silencing page](https://www.sigmaaldrich.com/US/en/applications/genomics/gene-expression-and-silencing).   * * * ## Related Products Slide 1 of 17 1 of 4 [ \ Sigma-Aldrich \ B6650 \ X-GlcA](https://www.sigmaaldrich.com/US/en/product/sigma/b6650) Quick View [ \ Sigma-Aldrich \ B3928 \ Blue-White Select™ Screening Reagent](https://www.sigmaaldrich.com/US/en/product/sigma/b3928) Quick View [ \ Sigma-Aldrich \ B9146 \ 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside](https://www.sigmaaldrich.com/US/en/product/sigma/b9146) Quick View [ \ Sigma-Aldrich \ D9156 \ Deoxyribonucleic acid, single stranded from salmon testes](https://www.sigmaaldrich.com/US/en/product/sigma/d9156) Quick View [ \ Sigma-Aldrich \ D8418 \ Dimethyl sulfoxide](https://www.sigmaaldrich.com/US/en/product/sigma/d8418) Quick View [ \ Sigma-Aldrich \ YEAST1 \ Yeast Transformation Kit](https://www.sigmaaldrich.com/US/en/product/sigma/yeast1) Quick View [ \ Sigma-Aldrich \ S1797 \ SOC Medium](https://www.sigmaaldrich.com/US/en/product/sigma/s1797) Quick View [ \ Sigma-Aldrich \ L7533 \ LB Broth with agar (Lennox)](https://www.sigmaaldrich.com/US/en/product/sigma/l7533) Quick View [ \ Sigma-Aldrich \ I6758 \ IPTG](https://www.sigmaaldrich.com/US/en/product/sial/i6758) Quick View [ \ Roche \ APAI-RO \ Apa I](https://www.sigmaaldrich.com/US/en/product/roche/apairo) Quick View [ \ Roche \ NOTI-RO \ Not I](https://www.sigmaaldrich.com/US/en/product/roche/notiro) Quick View [ \ Roche \ 11417959001 \ SuRE/Cut™ Buffer A](https://www.sigmaaldrich.com/US/en/product/roche/11417959001) Quick View [ \ Roche \ XBAI-RO \ Xba I](https://www.sigmaaldrich.com/US/en/product/roche/xbairo) Quick View [ \ Roche \ XTGHP-RO \ X-tremeGENE™ HP DNA Transfection Reagent](https://www.sigmaaldrich.com/US/en/product/roche/xtghpro) Quick View [ \ Roche \ XTG360-RO \ X-tremeGENE™ 360 Transfection Reagent](https://www.sigmaaldrich.com/US/en/product/roche/xtg360ro) Quick View [ \ Roche \ XTG9-RO \ X-tremeGENE™ 9 DNA Transfection Reagent](https://www.sigmaaldrich.com/US/en/product/roche/xtg9ro) Quick View [ \ Sigma-Aldrich \ OGS626 \ pSF-CMV-NEO-NH2-PPT-3XFLAG](https://www.sigmaaldrich.com/US/en/product/sigma/ogs626) Quick View * * * ## Featured Categories [](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/biological-buffers) [Biological Buffers](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/biological-buffers) Our extensive portfolio of high-purity biological buffers in various formulation and packaging formats provide superior solution stability and pH control throughout your bioprocess workflow applications. [View Products](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/biological-buffers) [](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/agarose) [Agarose](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/agarose) We offer a wide range of agarose products with various gel strengths, melting temperatures, gelling temperatures, and electroendosmosis (EEO) levels to support your research applications. [View Products](https://www.sigmaaldrich.com/US/en/products/chemistry-and-biochemicals/biochemicals/agarose) * * * Overview Related Articles & Protocols Support ## Protein Expression Vectors Plasmids, or protein expression vectors, are circular pieces of DNA that contain numerous functional elements for cloning and expressing the protein of interest. The selection of the appropriate expression vector is partially determined by the type of protein, the organism that will express the protein, and the specific application and scientific questions the researcher aims to address. The wide variety of expression vectors available allows researchers to choose which cloning, clone selection, and protein expression elements are best for their study. Once the gene sequence of interest and the expression vector is selected, researchers commonly use nucleases to precisely cut the vector at specific sites to allow the genetic sequence of interest to be cloned in-frame and ultimately expressed in a host organism. Importantly, researchers will commonly use chemically competent cells and bacterial transformation to host the recombinant DNA plasmids as they are stable when stored frozen at -70 °C. ## How to Transfect Cells Cell transfection refers to the process of introducing DNA or RNA, or proteins into eukaryotic cells. A variety of physical, chemical, and biological technologies are available to researchers to accomplish this part of the protein expression workflow. Physical methods, such as electroporation, or chemical methods, including calcium phosphate (CaPi), polyethylenimine (PEI), and lipofection reagents are all commonly used by researchers. Viral transduction systems, including lentiviral, oncoretroviral, and adenoviral are also available to scientists. However, the use of viral delivery methods often requires additional containment and monitoring for biosafety reasons. ## Protein Expression Systems Various organisms are used by researchers to express their protein of interest. Bacteria are commonly used by researchers as they efficiently uptake expression vectors, have fast doubling times, and can produce high quantities of recombinant protein under optimal conditions. However, as bacteria are prokaryotic organisms, they are not able to provide protein post-translational modifications that some proteins require. Eukaryotic cells, including yeast and mammalian cell lines, facilitate protein post-translational modification(s) and are also commonly used by researchers. The choice of the best cell line for recombinant protein expression and the protein expression strategy is highly dependent on the specific application needs and questions the scientist aims to address. [](https://www.sigmaaldrich.com/documents-search) [Looking for More Specific Information?](https://www.sigmaaldrich.com/documents-search) Visit our document search for data sheets, certificates and technical documentation. [Find Documents](https://www.sigmaaldrich.com/documents-search) ## Related Articles - [Restriction Endonucleases - The Molecular Scissors](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/restriction-endonucleases-the-molecular-scissors) The term “Restriction enzyme” originated from the studies of Enterobacteria phage λ (lambda phage) in the laboratories of Werner Arber and Matthew Meselson. - [Cloning Genes-of-Interest into a Plasmid Vector](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/restriction-enzyme-cloning-manual-cloning) This cloning protocol includes selecting the cloning system and plasmid vector, plasmid restriction digestion, fragment restriction digestion, gel excision, dephosphorylating DNA and more. - [Introduction to Cell Transfection](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/transfection-reagents) Transfection introduces genetic material into cells, aiding research in gene expression and cell biology. - [3xFLAG® System Expression Vectors for Ultra-Sens](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/3x-flag) The 3xFLAG system enhances fusion protein detection, offering improved sensitivity over traditional methods. - [SIG10 Chemically Competent Cells](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/cloning-and-expression/sig10-chemically-competent-cells) For best results, ligation reactions must be heat inactivated at 70º C for 15 minutes before transformation. Alternately, the reactions may be purified. - [See All (25)](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) ## Related Protocols - [Restriction Enzyme Digest Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/cloning-and-expression/restriction-enzyme-digest-protocol) Restriction enzyme collection has been optimized for digestion using five unique buffers. - [pGEX Vectors](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/cloning-and-expression/pgex-vectors) This page describes the characteristics of pGEX expression vectors used with Cytiva products. - [DNA Ligation Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/cloning-and-expression/dna-ligation-kit) Cloning process, joining linear DNA into a vector, is crucial for biotechnological experiments, enabling DNA fragment recombinant technology. - [Competent Cell Protocols](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/microbial-cell-culture/competent-cells) Technical Article on competent cells. Transformation is a process by which some bacteria take up foreign genetic material (naked DNA) from the environment. - [Dephosphorylation Procedures for DNA and Proteins](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/cloning-and-expression/dephosphorylation) Dephosphorylation of DNA using Calf Intestinal Alkaline Phosphatase, Shrimp Alkaline Phospha, Bovine Intestinal Alkaline Phosphatase - [See All (31)](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) ### Find More Articles and Protocols Enter Keywords Search ## How Can We Help In case of any questions, please submit a [customer support request](https://www.sigmaaldrich.com/US/en/support/customer-support) or talk to our customer service team: Email [custserv@sial.com](mailto:custserv@sial.com) or call +1 (800) 244-1173 ## Additional Support - [Calculators & Apps](https://www.sigmaaldrich.com/US/en/support/calculators-and-apps) Web Toolbox - science research tools and resources for analytical chemistry, life science, chemical synthesis and materials science. - [Customer Support Request](https://www.sigmaaldrich.com/US/en/support/customer-support) Customer support including help with orders, products, accounts, and website technical issues. - [FAQ](https://maestro.my.site.com/knowledgeportal/s/) Explore our Frequently Asked Questions for answers to commonly asked questions about our products and services. * * * __Featured Articles__ [Blue-White Screening & Protocols for Colony Selection](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/blue-white-screening) Learn about and find protocols for the blue white screen technique used in molecular biology research to identify recombinant bacterial clones for further analysis. [Restriction Endonucleases - The Molecular Scissors](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/restriction-endonucleases-the-molecular-scissors) The term “Restriction enzyme” originated from the studies of Enterobacteria phage λ (lambda phage) in the laboratories of Werner Arber and Matthew Meselson. [Bacterial Transformation Protocols](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/competent-cells) General protocols for growth of competent cells and their transformation (uptake of DNA). [T7 Promoter System](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/cloning-and-expression/t7-promoter-system) The T7 promoter is commonly used to regulate gene expression of recombinant proteins, which can be subsequently used for a variety of downstream research applications. 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