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A rapid microwell fluorescence immunoassay for cellular protein detection.

Lavigne C, Guignée de A, Thierry AR - Biol Proced Online (2008)

Bottom Line: In order to illustrate this method, we report on the detection of two different proteins, the cell cycle proteins cyclin D1 and p21(CIP1/WAF1), in untreated and 2-cyclopenten-1-one treated breast cancer cells.When the FICP method was compared with Western blot procedure, FICP was found to be superior for many characteristics.By using this method, we were able to quantify biological effects of a specific compound on protein levels in non-lysed cells and perform statistical analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beausejour Medical Research Institute, Moncton, New Brunswick, Canada. lavigne@phac-aspc.gc.ca

ABSTRACT
In this paper, we describe a simple, rapid, specific, sensitive, and reliable method, the FICP method (Fluorescence Immunoassay for Cellular Protein detection) which is readily applicable to the detection of proteins directly on cells cultured in 96-well plates. In order to illustrate this method, we report on the detection of two different proteins, the cell cycle proteins cyclin D1 and p21(CIP1/WAF1), in untreated and 2-cyclopenten-1-one treated breast cancer cells. When the FICP method was compared with Western blot procedure, FICP was found to be superior for many characteristics. By using this method, we were able to quantify biological effects of a specific compound on protein levels in non-lysed cells and perform statistical analysis. Therefore, we believe this screening assay could be very useful for detecting poorly expressed proteins and for drug development.

No MeSH data available.


Related in: MedlinePlus

Inhibition of cyclin D1 protein expression by cyclopentenone.MCF-7 cells exposed to cyclopentenone and untreated cells were processed after 4 hours of treatment. (A) Amount of cyclin D1 protein in each microwell was detected by immunofluorescence. Results are representative of four separate experiments, each done in 6 replicates. Data shown represent the mean of at least 5 replicates ± SD from one experiment. (B) CyclinD1 protein detection by Western blotting. Wells 1, 2, 3, and 4 represent cells treated with 150 ug/mL, 300 ug/mL, 600 ug/mL, or 1200ug/mL of cyclopentenone, respectively. Well C represents untreated control cultures. Results are representative of the amount of protein obtained in three separate experiments, and represent a single immunoblot. * p< 0.05, ** p< 0.001.
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Figure 02: Inhibition of cyclin D1 protein expression by cyclopentenone.MCF-7 cells exposed to cyclopentenone and untreated cells were processed after 4 hours of treatment. (A) Amount of cyclin D1 protein in each microwell was detected by immunofluorescence. Results are representative of four separate experiments, each done in 6 replicates. Data shown represent the mean of at least 5 replicates ± SD from one experiment. (B) CyclinD1 protein detection by Western blotting. Wells 1, 2, 3, and 4 represent cells treated with 150 ug/mL, 300 ug/mL, 600 ug/mL, or 1200ug/mL of cyclopentenone, respectively. Well C represents untreated control cultures. Results are representative of the amount of protein obtained in three separate experiments, and represent a single immunoblot. * p< 0.05, ** p< 0.001.

Mentions: 2-Cydopenten-l-one is known to cause cell cycle arrest in G1 and down-regulate cydin D1 protein expression in MCF-7 cells (19, 23). To determine whether the fluorescence immunoassay FICP was able to specifically deted a variation in the amount of cyclin D1 protein we treated the cells with different concentrations of cydopentenone for 4 hours. In these experiments, MCF-7 cells were plated in 96-well microplates at a density of 10,000 cells/well or in 75 cm2 flasks at a concentration of 10 x 106 cells/flask. After 24 hours plating, the culture medium was replaced by fresh medium supplemented with cydopentenone at a final concentration varying from 150 to 1200 /jg/mL. After 4 hours incubation, cydin D1 expression was measured by either the fluorescence immunoassay or Western Blot as described in Materials and Methods. The results are summarized in Fig. 2. The amount of cydin D1 protein detected by the fluorescence signal inversely decreased with the concentration of cydopentenone. At cydopentenone concentrations higher than 150 /jg/mL, the amount of cydin D1 protein was significantly decreased compared to control cultures, with maximum inhibition at 600 /ag/mL (p<0.05 for 300 /ag/mL and p<0.001 for 600 and 1200 /jg/mL, Fig. 2A). Results obtained by immunoblot analysis showed a diminution in expression of cyclin D1 protein in cells treated with cydopentenone when compared to untreated control cells (Fig. 2B). Detedion of cydin D1 protein was clearly visible in untreated control cultures however no cyclin D1 bands were detected in cells treated with 150 /jg/mL and higher concentrations of cydopentenone. The intensity of the tubulin signal decreased at increasing concentrations of cydopentenone probably due to the increasing cytotoxicity at doses higher than 300 /Jg/mL of cydopentenone.


A rapid microwell fluorescence immunoassay for cellular protein detection.

Lavigne C, Guignée de A, Thierry AR - Biol Proced Online (2008)

Inhibition of cyclin D1 protein expression by cyclopentenone.MCF-7 cells exposed to cyclopentenone and untreated cells were processed after 4 hours of treatment. (A) Amount of cyclin D1 protein in each microwell was detected by immunofluorescence. Results are representative of four separate experiments, each done in 6 replicates. Data shown represent the mean of at least 5 replicates ± SD from one experiment. (B) CyclinD1 protein detection by Western blotting. Wells 1, 2, 3, and 4 represent cells treated with 150 ug/mL, 300 ug/mL, 600 ug/mL, or 1200ug/mL of cyclopentenone, respectively. Well C represents untreated control cultures. Results are representative of the amount of protein obtained in three separate experiments, and represent a single immunoblot. * p< 0.05, ** p< 0.001.
© Copyright Policy - open acces
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683549&req=5

Figure 02: Inhibition of cyclin D1 protein expression by cyclopentenone.MCF-7 cells exposed to cyclopentenone and untreated cells were processed after 4 hours of treatment. (A) Amount of cyclin D1 protein in each microwell was detected by immunofluorescence. Results are representative of four separate experiments, each done in 6 replicates. Data shown represent the mean of at least 5 replicates ± SD from one experiment. (B) CyclinD1 protein detection by Western blotting. Wells 1, 2, 3, and 4 represent cells treated with 150 ug/mL, 300 ug/mL, 600 ug/mL, or 1200ug/mL of cyclopentenone, respectively. Well C represents untreated control cultures. Results are representative of the amount of protein obtained in three separate experiments, and represent a single immunoblot. * p< 0.05, ** p< 0.001.
Mentions: 2-Cydopenten-l-one is known to cause cell cycle arrest in G1 and down-regulate cydin D1 protein expression in MCF-7 cells (19, 23). To determine whether the fluorescence immunoassay FICP was able to specifically deted a variation in the amount of cyclin D1 protein we treated the cells with different concentrations of cydopentenone for 4 hours. In these experiments, MCF-7 cells were plated in 96-well microplates at a density of 10,000 cells/well or in 75 cm2 flasks at a concentration of 10 x 106 cells/flask. After 24 hours plating, the culture medium was replaced by fresh medium supplemented with cydopentenone at a final concentration varying from 150 to 1200 /jg/mL. After 4 hours incubation, cydin D1 expression was measured by either the fluorescence immunoassay or Western Blot as described in Materials and Methods. The results are summarized in Fig. 2. The amount of cydin D1 protein detected by the fluorescence signal inversely decreased with the concentration of cydopentenone. At cydopentenone concentrations higher than 150 /jg/mL, the amount of cydin D1 protein was significantly decreased compared to control cultures, with maximum inhibition at 600 /ag/mL (p<0.05 for 300 /ag/mL and p<0.001 for 600 and 1200 /jg/mL, Fig. 2A). Results obtained by immunoblot analysis showed a diminution in expression of cyclin D1 protein in cells treated with cydopentenone when compared to untreated control cells (Fig. 2B). Detedion of cydin D1 protein was clearly visible in untreated control cultures however no cyclin D1 bands were detected in cells treated with 150 /jg/mL and higher concentrations of cydopentenone. The intensity of the tubulin signal decreased at increasing concentrations of cydopentenone probably due to the increasing cytotoxicity at doses higher than 300 /Jg/mL of cydopentenone.

Bottom Line: In order to illustrate this method, we report on the detection of two different proteins, the cell cycle proteins cyclin D1 and p21(CIP1/WAF1), in untreated and 2-cyclopenten-1-one treated breast cancer cells.When the FICP method was compared with Western blot procedure, FICP was found to be superior for many characteristics.By using this method, we were able to quantify biological effects of a specific compound on protein levels in non-lysed cells and perform statistical analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beausejour Medical Research Institute, Moncton, New Brunswick, Canada. lavigne@phac-aspc.gc.ca

ABSTRACT
In this paper, we describe a simple, rapid, specific, sensitive, and reliable method, the FICP method (Fluorescence Immunoassay for Cellular Protein detection) which is readily applicable to the detection of proteins directly on cells cultured in 96-well plates. In order to illustrate this method, we report on the detection of two different proteins, the cell cycle proteins cyclin D1 and p21(CIP1/WAF1), in untreated and 2-cyclopenten-1-one treated breast cancer cells. When the FICP method was compared with Western blot procedure, FICP was found to be superior for many characteristics. By using this method, we were able to quantify biological effects of a specific compound on protein levels in non-lysed cells and perform statistical analysis. Therefore, we believe this screening assay could be very useful for detecting poorly expressed proteins and for drug development.

No MeSH data available.


Related in: MedlinePlus