Protein Assay

Question 1

Known protein

Answer 1

BSA samples

Question 2

Dilutions

Answer 2

BSA diluted with PBS
1:2
Wells AB 2-10 (18 wells)

Question 3

Blank

Answer 3

Column 1

Avg subtracted from unknowns

Question 4

Unknown protein

Answer 4

Wells CD 2-6 (10 wells)

Question 5

Spectrophotometer

Answer 5

Read at OD595

Question 6

Standard curve

Answer 6

Plot OD vs Prot concentration (ug/ml)

BSA most common

Question 7

Copper Dependent Assays

Answer 7

Biuret, Lowry, and bicinchinoic acid (BCA) Assay.
The color in the BCA assay is the result of the formation of Cu2+ -protein complex under alkaline conditions, followed by the reduction of Cu2+ to Cu+. The amount of reduction to Cu+is proportional to the amount of protein present. It has been shown that cysteine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2+ to Cu+. The BCA forms a blue-purple complex with Cu+.

Question 8

Bradford Assay

Answer 8

Measures protein concentration
Inexpensive and relatively sensitive
Coomassie Blue dye binds to protein molecules in acid pH and shifts color spectrum visibility from 465 nm to 595 nm
The triphenylmethane group of Coomassie Blue binds to nonpolar structures in proteins, and the anion sulfonate groups interact with protein cationic side chains (arginine and lysine side chains).

Question 9

Problems w Prot assays

Answer 9

1. Detection Range and accuracy of protein assays. Most protein assays have a detection range between 20 ug/ml and 1000 ug/ml. If you know that your samples contain greater than 1000 ug/ml of protein, then it beneficial to dilute samples before testing for accuracy. If your ODs are above or below the limit of detection you cannot extrapolate. Therefore, the best you can report is values >1000 ug/ml or <20 ug/ml.
2. Reproducibility. There are protein-to-protein variations in the detection using differentmethods. Even with relatively pure protein preparations, the measurements can vary by as much as 20% between different types of protein assays. In addition, reproducibility with the same assay and same protein samples can be variable with the variability in reagents, buffers, equipment and technique. Therefore, a protein standard curve must be performed EVERY time you do an assay.
3. Detergents. Protein assays are commonly done in salt solution buffers but sometimes your protein preparation has interfering substances such as detergents. The presence of many common detergents (e.g. NP-40, Triton-X-100) can cause non-specific reactivity (background color change) especially in Coomassie blue-based assays. Therefore, blank samples, protein standard and unknowns should all be prepared in the same buffer so that you can be sure that any signal is only from the protein present and not background from detergent.
4. Preparations. It can often be difficult to get accurate protein concentration measurements when testing crude preparations such as food preparations or cellular/bacteria lysates. Thecrude preparations are often inaccurate if they obscure color changes or have large particulate.