Separation of Proteins Part II

180°

Agenda

Review Separation of Proteins Part I

What is SDS-PAGE and how does it work?

Western Blotting

Today’s procedure

Reminders

Gel loading demo

What did you do two weeks ago?

What two proteins did you separate?

What two types of chromatography did you use to separate the mixture of proteins?

What is SDS-PAGE

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis A method of separating proteins based on their

electrophoretic ability (length and mass-to-charge ratio)

What is SDS-PAGE used for? Determination of protein size Protein identification Quantifying proteins Blotting applications- Western blots

Preparing Samples for SDS-PAGE

Sample PreparationSample Buffer components:

Tris-Cl Buffer: maintains pH (~6.8) SDS: sodium dodecyl sulfate, denaturing anionic

detergent β-mercaptoethanol (BME): breaks disulfide bonds

YOU MUST WORK WITH BME IN THE HOOD!!! Glycerol: increases sample density Tracking dye (Bromophenol blue): helps track

progress of runAdd protein sample and heat buffer to 90°C to

activate SDS and BME

Why is BME important?

BME breaks the disulfide bonds, removing tertiary and quaternary structure

What would happen if you did not add BME to your sample?

Why is SDS important?

SDS coats protein backbone with a negative charge in a constant weight ratio of 1.4g SDS/ g proteinand linearizes the protein, removing secondary structure

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SDS-PAGE Gel Set up

The SDS-PAGE Gel

The gel is comprised of a gradient of polyacrylamide, which helps to separate molecules by their size

4 %

20%

Acr

ylam

ide

Smaller proteins fit through the pores and travel farther

Larger proteins are trapped bythe pores and travel less

How does SDS-PAGE work?

An electric field is applied to the gel, allowing proteins to migrate by charge.

In which direction do the proteins run and why?

If there are multiple bands in the same lane, how would you identify whetherit contains your protein of interest?

Western Blotting

A method of detecting specific proteins by transferring the proteins from a gel to PVDF membrane and tagging the protein of interest with antibodies

What is an antibody?

An antibody is a specialized immune protein that recognizes a specific target molecule

Today’s Procedure

Add 75μL of Peak

Fraction 1 to 15 μL of

sample buffer

• Repeat for Peak Fraction 2

Heat Sample in heating

block (90°C) for 4

minutes

Centrifuge at max

speed for 30 sec.

• Make sure that tubes all contain the same volume and to properly balance the centrifuge

Load samples

onto gel in the correct

order

Gel Loading Order

Please load 15μL of sample and 10μL of MW marker

E. coli Lysate

Today you will run lysate from E. coli that over-express β-Galactosidase These E. coli make an excess

amount of the enzyme that breaks down the sugar β-Galactose

E. coli are lysed to spill the contents of the cells When run on a gel, you will see

the total proteins expressed by the cell at the time of lysis

Over-expressed proteins, will appear darker than other bands on the gel

Western blotting allows for you to determine if your protein of interest is your lysate, when it is difficult to determine from the gel

Setting up your Western Blot

Today, you will set up your western blot sandwiches: An electric field will

be run through the blotting apparatus, which will transfer the negatively charged proteins from the gel to the PVDF membrane

Data Analysis

This Week: Determine the protein

concentration using the Beer Lambert Law

Next Week: Calculate Rf values:

Protein migration/dye front migration Measure from the bottom of the well

to the dye front/protein band Create a semi-log plot (use the

correct paper!) MW vs Rf values for MW marker bands Use standard curve to determine the

molecular weight of your peak fractions

Use the 2-cycle semi-log paper

Standard curve of molecular weights of proteins based on Rf

values

(g/mol)

Reminders

Gloves and gogglesBME is toxic

You will find sample buffer in the hood Please place all tips/tubes contaminated with sample buffer in the

appropriately labeled waste containersRemember the order in which you set up your western blotSAVE YOUR FRACTIONS FOR NEXT WEEKLab report for Parts II and III due week of 4/6Problem Set 3 due the week of 3/30

Next week, we will: Analyze the stained gel Continue the western blot Perform the Lowry Protein Assay (Separation Part III)

Reminders

Please remember to properly cite the documents used in your concluding statements/discussions

http://classguides.lib.uconn.edu/content.php? pid=352130&sid=3243107