Construction of a Standard Curve for the Determination of Protein Concentration

Topics: Serum albumin, Bovine serum albumin, ELISA Pages: 2 (640 words) Published: September 26, 2012
Construction of a Standard Curve for the Determination of Protein Concentration

09/13/12
PCB 3023-007

Results

Figure1: Standard curve. This graph was constructed from the fixed amounts of Bovine Serum Albumin (BSA). Varying amounts of BSA at a concentration of .5µ/µl were mixed with the water and 1mL of coomassie dye. The equation y=0.0259x +0.0511 represents the best fit line for a data set taken from µg of protein equals two to ten µg. Optical density (OD) was surveyed using a Mach V visual spectrophotometer at 595.

Worktable for Coomassie Assay
Tube #| Sample ID | Water (µl)| Sample (µl)| Protein (µg)| OD @ 595| 1| BLANK| 1000| 0| 0| 0|
2| STAN| 1000| 4| 2| 0.090|
3| STAN| 1000| 8| 4| 0.166|
4| STAN| 1000| 12| 6| 0.214|
5| STAN| 1000| 16| 8| 0.259|
6| STAN| 1000| 20| 10| 0.302|
7| 1:50| 1000| 10| 6.33| 0.215|
8| 1:100| 1000| 10| 1.58| 0.092|
9| 1:250| 1000| 10| 0.92| 0.075|
10| UK| 1000| 10| 39.9| 1.085|
Table 1: Worktable for Coomassie Assay. Tubes two through six were created adding the amount of sample in µl to the corresponding tubes along with 1000µL of water and 1mL of coomassie dye. These tubes were mixed and the optical density was surveyed using a Mach V visual spectrophotometer at 595. Tubes seven through ten were created by adding the same amount of an unknown (UK) sample (10µl), coomassie dye (1mL) and water (1000µL). Each tubes 10µl sample varied by the amount it was diluted as shown. These tubes were also mixed and the OD was surveyed at 595. The UK protein in µg was assessed by taken the equation y=0.0259x+.0511 (Fig. 1) and solving for x where y is the OD.

Calculation of UK
Samp ID| µl of UK Assayed| OD @ 595| µg UK protein| µg UK/µl(Diluted Sample)| Dilution Factor| UK Conc in µg/µl| 1:50| 10| .215| 6.33| .633| 50| 31.65|
1:100| 10| .092| 1.58| .158| 100| 15.80|
1:250| 10| .075| 0.92| .092| 250| 23.00|...
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