LigandLink™ Universal Protein Labeling

Why use LigandLink™?

• Label your protein in living cells – LigandLink Labels are cell permeable
• Spend less time cloning – rather than using multiple vectors, clone only once; obtain different functionalities using different LigandLink Labels
• Small label – LigandLink Labels are unlikely to alter the characteristics of your protein of interest

LigandLink™ applications

• Protein localization & trafficking
• Protein:protein interactions
• Protein capture

Pre-made translocation vectors

Signal transduction pathways typically involve the movement of proteins throughout the cell in response to activation by some particular stimulus. The LigandLink Universal Labeling technology is ideal for studying such translocation events because it makes it simple to label the proteins in vivo. To make it even easier, we have already cloned a number of transcription factors into the pLL-1 vector, such as NFκB p65, which are ready to transfect into the mammalian cell line of your choice.

Figure 4: The LigandLink pLL-1 vector.

User friendly vector

The LigandLink vector, pLL-1, was designed for convenience of use. It features a CMV promoter for high-level expression of eDHFR fusion proteins, with Kanamycin for selection of stable cell lines. The multiple cloning site (MCS) was designed to facilitate cloning, whatever method you use. In addition to many popular restriction sites, the MCS includes three blunt-cutting restriction enzymes toward the 3´ end, each in a different reading frame with the eDHFR gene. This makes possible a number of PCR and restriction enzyme cloning strategies.

Ideal for multi-image capture experiments

Analysis of co-repressor and co-activator interactions is an important part of understanding protein function. However, analyzing interactions across multiple proteins can be time consuming and expensive. This is because each interacting partner must either be cloned into a different FP fusion vector, or primary antibodies must be labeled with multiple dyes. To overcome this problem, LigandLink Label comes in multiple formats that can be easily interchanged, depending on the FP partner of the interacting protein of interest, which saves you time and money.

Use of green fluorescent protein (GFP) and its derivatives has become commonplace in modern cell biology. However, biologically fluorescent proteins (FPs) are limited by their inherent properties. For example, FPs have a relatively low quantum yield, and it is difficult to engineer their spectral properties to suit specific applications.

Fluorescent dyes are a potential alternative to biological FPs as they are available in a broad variety of formats and can be engineered easily to ensure desirable spectral properties. However, because fluorescent dyes are synthetic molecules, it has not been possible to use them as a general tool for labeling specific proteins within a cell.

The LigandLink™ method

In the LigandLink method, the gene of interest is cloned in frame with the gene for E. coli dihydrofolate reductase (eDHFR) in the pLL-1 vector (Figure 4). The vector is then transfected into mammalian cells and used to express fusion protein. Twenty-four hours after transfecting, the protein of interest can be labeled simply by adding the LigandLink Label of choice to the cell medium (Figure 1). Depending on the cell type and the characteristics of the label, cells can be imaged in as little as 10 minutes.

The eDHFR protein was chosen as a fusion partner because it is a relatively small, monomeric protein (18 kDa vs. 27 kDa for GFP) that has been shown to have a high affinity for the ligand trimethoprim (TMP). TMP binds with a high specificity to the E. coli form of DHFR (K_I = ~1 nm), and a substantially lower affinity for endogenous DHFR (K_I = ~4 µm). This is because TMP is an antibiotic that was designed to specifically inhibit the bacterial enzymes responsible for the production of folic acid while not interacting with mammalian proteins. The result is extremely low background, because there is minimal binding of LigandLink Labels to non-tagged mammalian proteins. Moreover, TMP can be derivatized to carry a number of tags without substantially altering its affinity and specificity for eDHFR.

Thus, the LigandLink Universal Labeling technology uses eDHFR and a number of TMP derivatives as a ligand-receptor pair to provide a variety of functionalities.1 Cell-permeable LigandLink Labels are available with red and green fluorescent dyes (Figures 2 & 3).

 

Figure 2: Labeling of nuclear-localized eDHFR by LigandLink Fluorescein.

A nuclear localization sequence was cloned into pLL-1 and transfected into CHO cells. Twenty-four hours post-transfection, 5 µM LigandLink Fluorescein was added to the cells; 10 minutes later, the above image was taken.

 

Figure 3: Membrane-localized eDHFR labeling by LigandLink Hexachlorofluorescein.

The myristoylation/palmitoylation sequence from Lyn was cloned into pLL-1 and transfected into CHO cells. Twenty-four hours post-transfection, 10 nM LigandLink Hexachlorofluorescein was added to the cells; 2 hours later, the above image was taken.

References

1. Miller, L.W. et al. (2005) Nature Methods 2(4): 255-257.

Contents & Storage

Each LigandLink Kit contains 20 µg of vector and 100 reactions of LigandLink Fluorescein Label. Each reaction of LigandLink Label is sufficient to label a single well of a 96-well plate. Store at 4°C. All reagents are guaranteed stable for 6 months when stored properly.