5LMS0001 Bioscience Research Methods Assignment - University of Hertfordshire, UK

Workshop: Experimental Design for Bradford Microtitre Assay

Aims - The aims of this workshop are:

- To design an assay to measure the amount of protein in a sample using small volumes;

- To initiate the design of an experiment to explore the effect of possible interference by detergents on a protein assay.

Learning outcomes - On completion of this workshop students will:

- know how to make up a set of standards from a stock solution in order to construct a calibration curve;

- understand the considerations and constraints when designing an assay using a microtitre plate;

- know how to design an experiment to explore the effect of detergents on a protein assay.

Experiment 1: Determination of the concentration of protein in a sample of cell lysate

The first part of your task is to design an experiment based on the Bradford assay to determine the total protein content of the cell lysate. You will only have small volumes of sample to work with. You should use the practical booklet and your practical write-up from the Level 4 module Practical and Transferable Skills as a source of information in order to prepare for the practical sessions.

You are supplied with the following:

96 well microtitre plate

Gilson pipettes and tips

eppendorf tubes

1 mg cm^-3 stock solution of bovine serum albumin (BSA)

Bradford reagent

distilled water

sample of cell lysate

microtitre plate-reader

Experiment 2: The effect of detergents on protein estimation

The second part of your task is to design and carry out an experiment to determine the effect of two different detergents (Triton X-100 and SDS) on the assay. Remember you will need to incorporate controls as part of your experimental design.

Useful information - In designing your experiments, you may find the following information useful:

- The concentration of proteins in a cell lysate will depend on cell type, cell density and the volume of lysis buffer used. It usually lies in the range 0 - 5 mg/ml.

- Each well in the microtitre plate has a capacity of 300 μl.

- Detergents can be used at concentrations up to 5% in the preparation of cell lysates.

Preparation for BRMP2

An electronic version of this pro forma is posted on StudyNet (Proforma A). It must be completed and a hard copy shown to a member of staff during the practical session to be signed off. Failure to do this will result in you losing 10% of the marks for BRMi.

1. Complete the table below to show how you will make up a set of BSA standards.

Add rows to the table according to how many standards you intend to make up.

1. Make a note of the dilutions of cell lysate you will use to ensure that one of your samples will lie in the standard curve.

2. Use the template (attached) to show what you will put in each well of the microtitre plate.

1. Write a brief description of your protocol for Experiment 1. This should be written as a set of instructions.

Preparation for BRMP3

This should be completed before the BRMP3 practical session. Failure to do this will result in you losing 10% of the marks for BRMi.

1. Construct a table to show how you will make up your samples for Experiment 2.

Add rows to the table according to how many detergent samples you intend to make up. (Note: the cells in the column for detergent 2 will be blank for the samples containing detergent 1 and vice versa.)

1. Write a brief description of your protocol for Experiment 2.

Attachment:- Bioscience Research Methods Assignment File.rar