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Simple, Sensitive, and Rapid Detection of FLAG -Tagged,,,Fusion Proteins


Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins

Detection of expressed tags made easy

Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins

Qiyi Xie Quinn Lu Tanya Hosfield Peter Pingerelli Weiping Yang
Stratagene

Stratagenes TagDetect FLAG Protein Dipstick is an easy-to-use assay kit for detecting FLAG epitope-tagged fusion proteins. To visualize results, the kit uses a colloidal-gold (CG) labeled anti-FLAG M2 monoclonal antibody in a dipstick format. This highly specific monoclonal antibody detects the FLAG epitope regardless of its location in fusion proteins, and it will not cross react with non-specific prokaryotic and eukaryotic proteins. It is sensitive and fast: Detect as little as 10 ng/ml of expressed FLAG-tagged fusion protein in just 10 minutes.

Epitope-tagging technology provides a powerful means to functionally analyze genes in eukaryotic cells. With this technique, a tag-specific antibody is used to target and analyze the fusion protein within the cell, without making a specific antibody to each new protein under study.1

Epitope tags are usually short polypeptides with defined sequences, which can be terminally or internally incorporated into the protein of interest. Sequences coding for common epitopes have been incorporated into a variety of cloning and expression vectors. Since the FLAG peptide (DYKDDDDK) is a commonly used epitope tag, Stratagene developed the TagDetect FLAG Protein Dipstick. It can be used to monitor expression of FLAG-tagged proteins in either prokaryotic or eukaryotic cells, to follow FLAG-tagged proteins during purifica- tion steps, and to identify proteins following immun oprecipitation. Furthermore, it is superior to other methods, such as Western blotting; while Westerns require 100 ng/ml to 100 g/ml of tagged protein, Stratagenes assay detects as little as 10 ng/ml. The TagDetect FLAG Protein Detection Dipstick detects the FLAG epitope regardless of its location in fusion proteins.

A Fast, Easy Assay

Figure 1

A sample containing a FLAG-tagged protein is spotted onto the TagDetect strip membrane. Once the test strip is dipped into the running buffer, a complex forms containing the targeted fusion protein and the labeled antibody. Because the spotted sample is immobilized on the membrane, the formed complex appears as a red dot. Results can be obtained within 10 minutes (Figure 1 and Figure 2).

Figure 2

Sensitive and Specific

Figure 3

To determine the kits sensitivity, we used a GST-FLAG-calmodulin fusion protein purified from Schizosaccharomyces pombe as the test sample. As shown in Figure 3, as low as 10 ng/ml of the fusion protein can be detected. We further used S. pombe lysates to detect a GST-FLAG-MEK1 fusion protein expressed under the control of the inducible promoter.2 When samples derived from cells were grown under inducible conditions and prepared at a concentration of 40 g/ml of total protein, we observed a strong positive signal on the test strip. However, samples derived from cells grown under repressed conditions emitted no signal. When samples containing FLAG-tagged fusion proteins were expressed in E. coli, positive res ults were readily detected (Figure 4, strip #4). Thus, the TagDetect FLAG Protein Dipstick test strips can be used to monitor FLAG fusion gene expression and follow fusion proteins through purification steps and further analysis.

Figure 4

We carried out specificity studies by applying samples of nonrelevant cell extracts and purified proteins onto the membrane in the same manner as the positive samples. These samples were prepared from E. coli, yeast, and mammalian cells. The sample quantities and test procedures used were identical to the positive samples. At the end of the experimental time frame, the samples tested negative (Figure 4, strips #5 to #11).

Detecting FLAG Fusion Proteins in Mammalian Cell Lysates

To demonstrate how the TagDetect system works to monitor expression and purification, we used test strips to detect FLAG fusion proteins in mammalian cell lysates. Unlike the E. coli and the yeast samples described above, relative concentrations of the FLAG fusion protein are much lower in samples derived from mammalian epitope tagging.3 As shown in Figure 3, we detected a FLAG-luciferase fusion protein expressed from the CMV promoter in CHO cell lysates (strip #9) but did not observe them in the nonexpressed and nontagged proteins (strips #10 and #11). Thus, the kit provides a quick assay for FLAG fusion proteins before a full experimental protocol can be performed.

Conclusions

Stratagenes TagDetect assay kit detects and monitors FLAG-tagged fusion protein expression in yeast, E. coli, and mammalian cells. Its easy, unique technique speeds up detection, so the fastest possible results are realized, even with very low protein concentrations.

REFERENCES
  1. Kolodziej, P. A. and Young, R. A. (1991) Methods Enzymol. 194: 508-519.

  2. Lu, Q., et al. (1997) Strategies 10 (1): 4.

  3. Hosfield, T. and Lu, Q. (1997) Strategies 10 (3): 116-118.

The FLAG Technology is under license from Sigma-Aldrich Co.


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