Limits...
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

Activation of p21clpl/WAF1 protein expression in MCF-7 cells by cyclopentenone.Untreated and treated cells were processed after 4 hours of treatment and amount of p21clpl/WAF1 protein in each microwell was detected by immunofluorescence. Results are representative of three separate experiments done in triplicate. Each bar represents the mean of at least 3 replicates ± SD from one experiment. * p < 0.01.
© Copyright Policy - open acces
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2683549&req=5

Figure 04: Activation of p21clpl/WAF1 protein expression in MCF-7 cells by cyclopentenone.Untreated and treated cells were processed after 4 hours of treatment and amount of p21clpl/WAF1 protein in each microwell was detected by immunofluorescence. Results are representative of three separate experiments done in triplicate. Each bar represents the mean of at least 3 replicates ± SD from one experiment. * p < 0.01.

Mentions: To evaluate the specificity of the FICP assay, we next evaluated the effect of cyclopentenone on the expression of the cydin dependent kinase (CDK) p21CIP1/WAF1 protein. This protein was chosen, as the treatment with cydopentenone has resulted in an indudion of its protein expression in tumor (17-19). Therefore, we repeated the experiment as described above, and investigated the amount of p2lCIP1/WAF1 protein in parallel with those of cydin D1 protein. The indudion of p21CIF1/WAF1 and cydin D1 proteins were evaluated in separate wells of the same microplate. Results showed that a significant increase (p<0.01) in the amount of p21CIF1/WAF1 protein was observed in cells treated with 300 and 600 /ag/mL of cydopentenone compared to untreated control cells (Fig. 4). At higher doses, such as 1200 /jg/mL, amount of p21 decreases apparently due to toxidty of the compound.


A rapid microwell fluorescence immunoassay for cellular protein detection.

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

Activation of p21clpl/WAF1 protein expression in MCF-7 cells by cyclopentenone.Untreated and treated cells were processed after 4 hours of treatment and amount of p21clpl/WAF1 protein in each microwell was detected by immunofluorescence. Results are representative of three separate experiments done in triplicate. Each bar represents the mean of at least 3 replicates ± SD from one experiment. * p < 0.01.
© Copyright Policy - open acces
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683549&req=5

Figure 04: Activation of p21clpl/WAF1 protein expression in MCF-7 cells by cyclopentenone.Untreated and treated cells were processed after 4 hours of treatment and amount of p21clpl/WAF1 protein in each microwell was detected by immunofluorescence. Results are representative of three separate experiments done in triplicate. Each bar represents the mean of at least 3 replicates ± SD from one experiment. * p < 0.01.
Mentions: To evaluate the specificity of the FICP assay, we next evaluated the effect of cyclopentenone on the expression of the cydin dependent kinase (CDK) p21CIP1/WAF1 protein. This protein was chosen, as the treatment with cydopentenone has resulted in an indudion of its protein expression in tumor (17-19). Therefore, we repeated the experiment as described above, and investigated the amount of p2lCIP1/WAF1 protein in parallel with those of cydin D1 protein. The indudion of p21CIF1/WAF1 and cydin D1 proteins were evaluated in separate wells of the same microplate. Results showed that a significant increase (p<0.01) in the amount of p21CIF1/WAF1 protein was observed in cells treated with 300 and 600 /ag/mL of cydopentenone compared to untreated control cells (Fig. 4). At higher doses, such as 1200 /jg/mL, amount of p21 decreases apparently due to toxidty of the compound.

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