Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Mesenchymal stem cell)
Specification
Mesenchymal stem cell
Fixative
Paraformaldehyde
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2%
Other product details
Dilution
1/200
Incubation time
8 hour(s) and 0 minute(s)
Secondary antibody
Secondary antibody
None used
Additional data
Additional Notes
Epitope tags is a molecular reporter system used to localize gene products in a variety of living cells, to identify the cellular localization of the associated tagged proteins of interest, to track the translocation of tagged proteins within a subcellular compartments, and, to characterize new genes without creating protein-specific antibodies against the gene/protein of interest. Moreover, as they are very small in size; epitope tags typically do not alter the functional properties of tagged protein. In addition, uses of epitope tagging system in molecular biology have great advantage due to wide availability of the tag-specific generic antibodies.
Therefore, a recombinant DNA technology is used in our experiment to construct a fusion protein which consists of a gene of our interest (SDF-1) and an epitope tag (Myc) that fused to the carboxy terminal of the targeted protein (SDF-1). Lentivirus, carrying the targeted DNA (SDF-1, MYC), driven by a CMV promoter has been transfected into cultured rat mesenchymal stem cells (MSC). We then used immunofluorescence staining using Myc-Tag polyclonal antibody conjugated with FITC to detect and localize the transfected proteins (SDF-1) that containing the Myc epitope tag in MSC.
The cultured MSC has been fixed with 2% paraformaldehyde for 10 min, then permealized the cells with Triton-X100 (0.1%) for 10 min and incubated cells with anti-Myc primary antibody (1:200 dilution) for overnight at 4 degree centigrade. The image has been taken using a regular fluorescence microscope. Image here demonstrate Myc (Green, that shows both nuclear and cytoplasmic localization in MSC, upper left panel) and DAPI (blue-nuclear counterstain, upper right panel), overlay image (lower left panel) and image of the delineated area (lower right panel).
This is important to note that, contrary to the original vendor data sheet (where it has been mentioned regarding the localization of the Myc, as strictly nuclear), here, our data shows Myc could be present in both cytoplasm and nucleus depending on the localization of the tagged protein used in the experiment. There are several evidences in the literature that SDF-1 is usually present in both nuclear and cytosolic compartment of the cells and our data is consisted with those results.
Overall, this data provides a comprehensive idea of Myc distribution in cell. Furthermore, these results show that the Myc- tag does not alter the pattern of normal SDF-1 localization and this outcome may be suggestive of the preserved biochemical properties of the SDF-1 following Myc-tag. Lastly, the image here clearly depict that Myc-tag (here, the transfection efficacy is about 70-75%) could be used to quantify the expression level of the protein of interest, when DAPI is used in conjunction to identify the total number of cell nuclei present in the field.
Therefore, a recombinant DNA technology is used in our experiment to construct a fusion protein which consists of a gene of our interest (SDF-1) and an epitope tag (Myc) that fused to the carboxy terminal of the targeted protein (SDF-1). Lentivirus, carrying the targeted DNA (SDF-1, MYC), driven by a CMV promoter has been transfected into cultured rat mesenchymal stem cells (MSC). We then used immunofluorescence staining using Myc-Tag polyclonal antibody conjugated with FITC to detect and localize the transfected proteins (SDF-1) that containing the Myc epitope tag in MSC.
The cultured MSC has been fixed with 2% paraformaldehyde for 10 min, then permealized the cells with Triton-X100 (0.1%) for 10 min and incubated cells with anti-Myc primary antibody (1:200 dilution) for overnight at 4 degree centigrade. The image has been taken using a regular fluorescence microscope. Image here demonstrate Myc (Green, that shows both nuclear and cytoplasmic localization in MSC, upper left panel) and DAPI (blue-nuclear counterstain, upper right panel), overlay image (lower left panel) and image of the delineated area (lower right panel).
This is important to note that, contrary to the original vendor data sheet (where it has been mentioned regarding the localization of the Myc, as strictly nuclear), here, our data shows Myc could be present in both cytoplasm and nucleus depending on the localization of the tagged protein used in the experiment. There are several evidences in the literature that SDF-1 is usually present in both nuclear and cytosolic compartment of the cells and our data is consisted with those results.
Overall, this data provides a comprehensive idea of Myc distribution in cell. Furthermore, these results show that the Myc- tag does not alter the pattern of normal SDF-1 localization and this outcome may be suggestive of the preserved biochemical properties of the SDF-1 following Myc-tag. Lastly, the image here clearly depict that Myc-tag (here, the transfection efficacy is about 70-75%) could be used to quantify the expression level of the protein of interest, when DAPI is used in conjunction to identify the total number of cell nuclei present in the field.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Dr. Mal Niladri
Verified customer
提交于 Nov 08 2006