Bios 211 Pre-Lab #4

Honor code policy

Since you have worked with amounts, volumes, and concentrations for two weeks, you should be prepared to do these calculations on your own. Please complete this prelab without any outside help, including TAs, instructor, or fellow students. You may, of course, consult the course web site and other references.

Part 1: Sample preparation for SDS-PAGE

REMINDER: for all pre-labs you may use the <tab> key to move from one response field to another; do not hit <return> or <enter>, however, as doing so immediately submits the form.

Your responses are to reflect what you will do in the laboratory; these are to be the amounts and volumes that you will actually use.

Characterization of membrane proteins by SDS-PAGE requires denaturation of protein samples by sodium dodecyl sulfate and a reducing agent in a buffer containing tris base, chloride anions, glycerol, and a tracking dye. In order to load sufficient protein in a manageable volume, concentrations of prepared samples must be uniform. This prelab focuses on how to mix samples for electrophoresis to an appropriate final concentration.

Objectives

1. Calculate the volumes of sample, water, and 2x concentrated sample buffer that you will mix up in lab, to prepare each of your aliquots for SDS-PAGE
2. Write a brief description of how to prepare any sample in order to dilute it to an appropriate concentration in sample buffer for SDS-PAGE. That is, write a general methodology.

Information needed

• All sample buffer components (SDS, glycerol, tris, DTT, etc.) must be at full strength during the denaturing process and must remain at full strength when the samples are loaded into the gels
• You need the concentration of each of your samples, from last week
• Each well of a typical 'mini-gel' holds 20 µl of sample
• Good results are obtained when an amount of 40 µg protein is loaded in each well
• To ensure a uniform electric field, all wells should contain the same volume of sample
• The sample buffer, containing all components except the sample, has been prepared as a 2x concentrated stock solution
• Final concentration of sample buffer must be 1x
• You will need to load at least two wells with each sample, and there may be some losses while transferring denatured samples - therefore you should prepare an excess volume
• It is acceptable to load less protein if the sample concentration is too low to start with

Complications

• Some samples may contain too little protein in order to meet the above criteria — if that is the case, then simply dilute 1:1 with 2x concentrated sample buffer
• Some samples are very concentrated to start with, and will require considerable dilution
• Our pipettors are accurate down to a minimum of 2 µl. We can't accurately pipet a volume of < 2 µl.

Resources

In addition to the web pages describing the study itself (SDS-PAGE), you will need to recall your expertise with solutions, dilutions, and stock solutions. You will find the sample preparation much easier to do if you treat both your 2x concentrated sample buffer and your protein samples as stock solutions to be incorporated into the same final "working solution." An example of this type of dilution, called "working with samples," is available on the page titled "working with stock solutions."

Assignment

1. Prepare a table of exact volumes of sample, water, and 2x concentration sample buffer that you will mix for each of your aliquots. Enter the information below.

Units

Please write out the full names of all units including prefixes (for example, "microliters," not "µl," and "milligrams," not "mg"). Report the units you are using for volume and concentration. Enter only numbers for the remaining answers.
Volume:
Concentration:

Plasma fraction (typically 4 to 10 mg/ml)

Initial concentration (from last week)
Volume of sample, water or buffer, and 2x concentrated sample buffer, respectively , ,

Lysate fraction

Initial concentration (from last week)
Volume of sample, water or buffer, and 2x concentrated sample buffer, respectively , ,

Cytosol fraction

Initial concentration (from last week)
Volume of sample, water or buffer, and 2x concentrated sample buffer, respectively , ,

Membrane fraction

Initial concentration (from last week)
Volume of sample, water or buffer, and 2x concentrated sample buffer, respectively , ,

Part 2:

Question 1

The Bradford assay for a protein sample gave an absorbance of 0.64. From a standard curve, the absorbance reading corresponded to 81 micrograms of protein. The volume of sample was 23 microliters. The concentration of protein in the sample should be reported as

Question 2

Please select from the list the specific procedures that you will use in the preparation of a sample for SDS-PAGE, and place them in the proper order by letter (example: EDABC). Please don't include punctuation, spaces, or comments – just the letters.

A) heat the sample
B) mix with denaturing buffer including sodium dodecyl sulfate, DTT, etc.
C) dilute the sample with a protein standard
D) centrifuge the sample
E) treat the sample with an oxidizing agent
F) dilute sample to standard concentration and volume

Question 3

What toxic substance will be used when you conduct SDS-PAGE on protein samples?

What precautions must be taken when working with the substance (up to five words per item)?

Question 4

Name two things that can be learned about proteins by conducting SDS-PAGE.

#1

#2

Question 5

In electrophoresis, what force drives proteins through a gel?

***DON'T FORGET TO ENTER YOUR NAME***

You should print off and/or save the completed form to facilitate going over instructor's comments later.

First name:

Last name:

Print off the "Form Submitted" page if possible, and as usual bring it to lab. When the page comes up, double check that your name is there.

— Thank you! —