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- Protocol: transfection of cerebellar slice cultures with Xfect reagent
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FAQs about the Xfect Protein Transfection Reagent
Rapid, high-efﬁciency, low-toxicity protein transfection
- Transfect a large amount of active protein
- Virtually no cytotoxicity, unlike lipofection
- Very high efﬁciency, even in stem or hematopoietic cells
- Simple protocol—assay for your protein in just 2 hours
The Xfect Protein Transfection Reagent uses a cell-penetrating peptide developed at Takara Bio to bind and transport active proteins directly into a wide variety of mammalian cell types, including hard-to-transfect human suspension cell lines and mouse embryonic stem cells.
What is Xfect Protein Transfection Reagent and how does it work?
Xfect Protein Transfection Reagent is a modiﬁed peptide with a cell-penetrating activity whose amino acid composition enables it to interact with a protein cargo and transport this protein across the cell membrane barrier. Just incubate your protein of interest with Xfect in the supplied buffer for 30 min, then apply the mixture to your cells—2 hours later, you are ready to assay for protein activity.
Why transfect puriﬁed proteins directly?
Protein transfection is extremely rapid compared to traditional gene expression studies using transfected DNA (1–2 hours compared to 18–48 hours) because it bypasses the cellular processes of transcription and translation. It also facilitates studies involving transient effects of proteins, and avoids potentially harmful, random DNA integration into the genome of the target cells. Xfect Protein Transfection Reagent makes it possible to deliver active proteins directly into cells for studies that involve transcriptional regulation, the cell cycle, apoptosis, oncogenesis, epigenetics, cell regeneration, and transdifferentiation.
Martin, S. J. et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545–56 (1995).
What are the advantages of Xfect Transfection Reagent compared to other protein delivery technologies?
The two technologies most frequently used for protein delivery are based on lipids and cell-penetrating peptides. Transfection with lipid-based reagents tends to result in high cytotoxicity, while peptide-based reagents transfect with low efficiency. Xfect Protein Transfection Reagent offers the best advantages of both (i.e., it retains low cytotoxicity and delivers more protein to a higher percentage of target cells). Moreover, this reagent can transfect cells that are growing at a higher density than competing products, which is important because assays are often performed within a few hours post-transfection, and the higher cell densities ensure sufficient material for downstream analysis.
Is the transfected protein active?
Yes, the protein is active. Assays that measure enzyme activity (such as caspase-3 or β-galactosidase) show that these proteins retain their activity after transfection with Xfect Protein Transfection Reagent, while fluorescent proteins retain their ability to fluoresce.
Does Xfect Protein Transfection Reagent deliver a high amount of protein?
Yes, Xfect Protein Transfection Reagent delivers far more protein per cell than the leading protein transfection reagent (Product C), a peptide-based reagent, in a broad range of cell types. This was demonstrated when we compared the amounts of β-galactosidase and fluorescent proteins delivered by each reagent. When we compared six different cell types, Xfect protein delivered 3- to 13-fold more protein than Product C. The biggest disadvantage of other protein delivery reagents is their limited ability to allow monitoring of a protein's biological effects (due to an inability to deliver sufficient active protein). Xfect protein delivers more protein per cell than other reagents, so it should expand the applications for protein delivery and/or allow you to use less protein for transfection.
Does Xfect Protein Transfection Reagent affect cell viability?
No, as demonstrated when we performed a WST-1 assay to compare the effects on HeLa cell proliferation of Xfect Protein Transfection Reagent and Product P (a popular lipid-based protein transfection technology). The Xfect reagent did not affect cell proliferation compared to untreated cells, while Product P decreased cell proliferation by more than 30%. When we used Xfect Protein Transfection Reagent to transfect DsRed-Express (a red ﬂuorescent protein) into a commonly used mouse embryonic stem cell line and measured cell viability using trypan blue dye exclusion, the cells retained 100% viability.
What is the transfection efﬁciency of Xfect Protein Transfection Reagent?
The transfection efﬁciency of Xfect Protein Transfection Reagent can vary depending on the cell type and protein delivered. However, in all cell lines tested, Xfect protein outperforms Product C. When we transfected AcGFP1 (green ﬂuorescent protein) into several different cell types using Xfect protein and measured transfection efﬁciency using ﬂow cytometry, more than 90% of the cells were transfected in four out of the eight cell types that we tested, and seven of the eight showed more than 80% transfection efﬁciency (Panel A). When immunoﬂuorescence microscopy was used to compare the efﬁciency of AcGFP1 transfection into HeLa cells using Xfect protein and Product C (a leading competitor), a high level of transfection was observed only for the cells treated with Xfect Protein Transfection Reagent (Panel B).
Will Xfect Protein Transfection Reagent work for all cell types, including cell types that cannot be easily transfected with DNA?
Target cell choices are not limited. We have successfully transfected a variety of mammalian cell types (human and rodent) with high efﬁciency, including hard-to-transfect human suspension cell lines (Jurkat, HL-60), which were transfected with close to 100% efﬁciency. Mouse ES cells (E14TG2A) were transfected at an amazing 87% efﬁciency when using Xfect Protein Transfection Reagent despite the fact that they cannot be transfected using Product C from a leading competitor (see "What is the transfection efficiency of Xfect Protein Transfection Reagent?" above for more details)
Does Xfect Protein Transfection Reagent work for all proteins?
Since every protein has unique physicochemical characteristics, it is unlikely that any protein transfection reagent will transfect all proteins. However, Xfect Protein Transfection Reagent has been used successfully to transfect numerous proteins with a wide range of molecular weights and physicochemical characteristics. Proteins we have successfully transfected include ß-galactosidase, ﬂuorescent proteins such as rAcGFP1 and rDsRed-Express, anti-alpha tubulin antibody, caspase-3, and R-phycoerythrin. R-phycoerythrin is a very large protein (~240 kDa). Optimizing the ratio of reagent to protein will allow you to determine the appropriate conditions for delivery of your protein of interest.
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