BIO2101

Comprehensive Biology Laboratory

Exercise #3b

PBS vs Water

 PBS (Phosphate Buffered Saline)

 For washing and dilution of cell culture

 Contains sodium chloride, sodium phosphate and potassium phosphate; pH = 7.4

 Osmotic pressure (isotonic)

 Lysis buffer (Triton X-100)

Triton X-100 is widely used to lyse cells to extract protein or organelles, or to permeabilize the membranes of living cells. It is a nonionic surfactant that has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon hydrophobic group, which disrupt the hydrophobic-hydrophilic interactions of membrane component.

Protein assay – Bradford reagent (contains phosphoric acid, avoid contact with the mouth and skin)

 Standard curve

Construction of protein standard plot

Prepare 1:1 serial dilutions of bovine serum albumin (BSA) standard (0.6 mg/mL) provided to construct a range of standard solutions between 0 and 0.6 mg/mL (e.g. 0, 0.0375, 0.075, 0.15, 0.3 and 0.6 mg/mL). This can be accomplished as follows:

To start serial dilution, pipette 50 µL of 0.6 mg/mL BSA standard from tube 6 into tube 5. Then pipette 50 µL from tube 5 into tube 4 until you have reached 0.0375 mg/mL conc. (tube 2). Do not dilute into tube 1 so that it can serve as a “Blank” for the assay.

Protein standard curve

Protein Standard plot

1. Linear Regression Straight line equation Y=aX+b

a is the slope, b is the y-intercept.

2. The coefficient of determination is such that 0 < r 2 < 1, and denotes the strength of the linear association between x and y.

The coefficient of determination represents the percent of the data that is the closest to the line of best fit.

3. Coefficient of determination (R2 ) should be 0.9 or above

Protein Assay

Lab report for Exercise 3

 Introduction

 Procedure (Materials and Methods)

 Results (used the data of cell numbers to 1. plot the curves of cell concentration; and the data of protein concentration to 2. plot a growth curve of the protein amount per cell)

 Discussion

Lab report #3 requirement (result)

Part A: Cell counting with the hemocytometer

◦ Data shown by table and graph for 4% and 20% FBS cell culturing conditions

◦ Table should contain data of i) raw cell count, ii) dilution factor (if any), iii) standard deviation and iv) average number of cells

◦ Formulae for calculating ‘Av. no. of cells’ from ‘Raw cell count’ and ‘dilution factor’ obtained from Part A

Lab report #3 requirement (results cont.)

◦ Graph: growth curves of cell concentration

◦ Data (Conc. of cells)