LABORATORY 5 - Western Oregon University



INTRODUCTION

In this lab you will use SDS PAGE (polyacrylamide gel electrophoresis) to analyze the proteins in Dictyostelium discoidium cells. SDS PAGE separates proteins on the basis of their molecular weights, and there are a variety of proteins in each of these types of cells. If we arbitrarily assume that there are ten different housekeeping proteins found in both wild type and mutant cells, and there are four proteins unique to wild type and six unique to the mutatnt cells, how many bands are expected in SDS gel patterns of each of type of cell?

On line simulation SDS gel electrophoresis



OBJECTIVES

1. Understand the theory of SDS PAGE.

You can be certain that you understand the theoretical basis of SDS gel electrophoresis if you can answer the following questions.

Why is there a separating gel and a stacking gel? What is the function of SDS? of the β-mercaptoethanol? of boiling the samples? What will be the relative position on the gel of large and small proteins? How will migration on the gel be affected by the % acrylamide? What corrections need to be made if the gel pattern displays “smearing”, “smiles”, no proteins, all the proteins at the dye front? Is it possible to recover active proteins from an SDS PAGE gel? How or why not?

2. Use SDS PAGE to determine the molecular weight of the proteins in a sample.

Your analysis of this experiment is complete after you have used the instructions on page 4, Section C: Analysis.

MATERIALS

sample from previous lab

boiling water bath with foam test tube rack

P10 micropipette & tips

β-mercaptoethanol

3x sample buffer

microcentrifuge

1 L DDH2O

PAGE apparatus with 1 separating gel & 1 sample comb/team, 1 gel chamber/2 teams & 1 power supply/6 teams

Reagents to prepare stacking gel

100 ml 5x running gel buffer

500 ml graduate

“hooked” pastuer pipette

P20 micropipette & tips

1 microwaveable dish & top/ 2 teams

microwave

100 ml Fairbanks A staining solution

100 ml Fairbanks D staining solution

PROCEDURES:

A. SDS-polyacrylamide gel sample preparation

1. Calculate the volume of SDS sample buffer you will need if you are going to add one volume of lysed cells to 4 volumes of sample buffer. Add 5 ul of β-mercaptoethanol to every 100 ul of SDS sample buffer that you will need and combine the cell prep and sample buffer.

2. Loosen the caps slightly so that they do not pop off when the tubes are heated. Load your micro tubes into a Styrofoam rack and place the rack in a boiling water bath so that the level of liquid in each tube is below the surface of the water. Boil the samples for five minutes.(The heat, in combination with the strong anionic detergent SDS and the reducing agent 2-mercaptoethanol, denatures the membrane proteins, unraveling their tertiary and secondary structures. In addition, the negatively charged SDS molecules "coat" protein surfaces, giving them a strongly negative net charge. As a result, the proteins will migrate through the pores in the polyacrylamide gel, from cathode to anode, primarily according to their molecular weights.)

3. Retrieve your samples from the boiling water bath and tighten the caps. Allow them to cool to room temperature before loading them onto the gel.

B. SDS-polyacrylamide gel preparation, electrophoresis and staining.

CAUTION: Acrylamide and bis-acrylamide are both neurotoxins and should be handled with extreme care! Always wear gloves when working with acrylamide or any solution containing it! Be sure that all acrylamide waste is placed in the proper receptacles.

1. Wash the glass plates and spacers with warm detergent solution, rinse well with distilled water, and give a final rinse with acetone.

2. Assemble the gel apparatus as instructed by the manufacturer.

3. At least four hours before the gel is to be run, mix the separating gel solution according to Table 1, selecting a polyacrylamide concentration consistent with the molecular weight range of the proteins you are concerned with separating. Combine all ingredients in the order given in the table, swirling the flask after each addition. Add the TEMED and mix gently. Immediately pour the separating gel to approximate total height of the gel comb below the top of the shorter of the glass plates.

Table 1. Preparation of SDS-polyacrylamide separating gel

(all volumes in ml needed to prepare 2 BioRad miniProteanII gels)

12% 10% 7.5%

|Molecular Weight Range |10-100,000 |20-175,000 |40-250,000 |

|Deionized water |3.35 |4.10 |4.85 |

|1.5 M Tris-HCl, pH = 8.8 |2.50 |2.50 |2.50 |

|Acrylamide stock |4.00 |3.25 |2.50 |

|10% SDS |0.10 |0.10 |0.10 |

|10% ammonium persulfate (freshly prepared) |0.050 |0.050 |0.050 |

|TEMED |0.005 |0.005 |0.005 |

.

4. Within two hours of running the gel, and after the separating gel has polymerized, mix 10 ml of the stacking gel solution according to Table 1. Combine all

ingredients in the order given in the table, swirling the flask after each addition. Add the TEMED and mix gently. Immediately pour the stacking gel to about 2 mm from the top of the glass plates.

Table 2. Preparation of 4% SDS-polyacrylamide stacking gel

(all volumes in ml needed to prepare 2 BioRad miniProteanII gels)

|Deionized water |3.05 |

|0.5M Tris-HCl, pH 6.8 |1.25 |

|10% SDS |0.05 |

|Acrylamide stock |0.67 |

|10% ammonium persulfate (freshly prepared) |0.025 |

|TEMED |0.005 |

5. Immediately insert the comb into the stacking gel mixture being careful not to trap any air bubbles under the teeth of the comb. Oxygen will inhibit the polymerization and distort the bottom of the sample well. Be sure the comb is centered. Allow the gel to polymerize for at least 15 minutes.

6. After polymerization is complete, carefully remove the comb from the stacking gel. Pull the comb up with a very slight side-to-side motion. Be careful not to disturb the well dividers. Fill each well with diluted running buffer.

7. Dilute 70 ml of 5X running buffer with 280 ml of deionized water for one electrophoresis run. Add running buffer to the upper chamber and the lower buffer chamber: Fill the lower chamber to slightly above the bottom of the running gel. Use the “hooked” Pasture pipette to remove any bubbles from beneath the bottom of the gel. The gel is now ready to be loaded with samples.

8. Load 10 ul of sample prepared as in Procedure Section B. into each well, using a predetermined and accurately recorded distribution of samples into the wells of the gel(s). Use a micropipettor with disposable tips, changing tips whenever changing from one sample to another. Alternatively, use a Hamilton syringe to load the samples, rinsing the Hamilton 8-10 times with distilled water after each use.

9. Electrophorese samples at 200v, constant voltage setting. No voltage adjustment is necessary for the thickness of the spacers or the number of gels. The usual run time is about 45 minutes.

10. When the tracking dye has reached the bottom of the gel, turn off the power supply and disconnect the power cables. Remove the lid and remove the gels.

l1. Disassemble the sandwiches (wear gloves) by gently prying the glass plates apart by twisting one of the spacers.

12. The gel will stick to one of the plates. Leave it there for now. Cut the stacking gel off with one of the spacers and discard the stacking gel. Notch the upper right hand corner of the gel (over the last well) so that you will know the proper orientation of the gel once it is removed. This is very important!!!

13. Use a Pasture pipette to squirt Fairbanks Staining Solution A between the gel and the glass plate; the gel will gradually slide off the plate. Let it gently fall into a microwaveable plastic dish with 100 ml of stain for every pair of gels

Cover the plastic dish loosely and microwave the gels in Fairbanks solution A

for 2 minutes at full power, or to just short of boiling

14. Rock the gels gently for 5 minutes at room temperature.

15. Decant the staining solution into a container in the hood – the fumes from the hot isopropyl alcohol and acetic acid are quite obnoxious – and rinse the gels twice with 100 ml of DDH2O at room temperature.

16. .Pour off the second DDH2O rinse, add 100 ml of Fairbanks Solution D, toss a crumpled Kimwipe into the dish, cover

loosely and microwave for 1 minute and 30 seconds at full power.

17. Cool for 5 minutes at room temperature with gentle shaking, discard the solution and store the gels in DDH2O until scanning them to produce a permanent record of the results.

18. Rinse plates, comb and spacers with distilled water and place each in the peg rack when you are done with them.

C. Analysis of SDS-polyacrylamide gels

1. Lay your stained gel on the CM Rule & Relative Mobility Calculator (to be handed out in lab) with the tracking dye on the bottom line of the Rule (Relative Mobility = 1.0). Position the gel so the top of the molecular weight standard lane is aligned with the top line of the Rule (Relative Mobility = 0). Read the Relative Mobility of each of the stained bands in the lane. Record these values and the molecular weight of each band in the Molecular Weight standards in the columns of the Data Sheet that correspond to the lane number in which the molecular weight standards were loaded.

2. Using the semilog paper (to be handed out in lab) you’ve been supplied, plot the relative mobility as a function of the log of the molecular weight for the proteins in the molecular weight standard sample. (Since the log scale is on the Y-axis, this graph does not conform to the convention of plotting the independent variable on the X-axis and the dependent variable on the Y-axis. For this graph, the dependent variable will be plotted on the X-axis and the independent variable will be plotted on the Y-axis.)

3. Now determine the Relative Mobility of each band in the gel lanes that contain your RBC and WBC samples and record this values in the appropriate columns of the Data Sheet. Using the standard curve constructed in steps 1 and 2, determine the molecular weights for each of these bands and enter the values in the Data Sheet.

4, the information from your gels by answering the following questions.

How many bands are visible in RBC and WBC samples?

What are the molecular weights of bands found in both the RBC and the WBC lanes? What are the molecular weights of the heaviest bands in each sample? Of the minor bands?

What are the molecular weights of bands unique to the RBC lane? To the WBC lane?

Is it possible to provisionally assign identities to any of the bands in either sample?

Are these assignments consistent with the major proteins that RBCs and WBCs should have to accomplish their unique functions?

REAGENTS AND BUFFERS

Sample Buffer, SDS Reducing Buffer: Store at RT.

|deionized water |3.8 ml |

|0.5 M Tris-HCl, pH = 6.8 |1.0 ml |

|Glycerol |0.8 ml |

|10 % sodium dodecyl sulfate (SDS) w/v |1.6 ml |

|2-mercaptoethanol |0.4 ml |

|1% (w/v) bromophenol Blue |0.4 ml |

Dilute the sample at least 1:4 with sample buffer, and heat to 95oC for 4 minutes.

1.5 M Tris-HCl, pH = 8.8:

|Tris Base/deionized water |27.23 g/~80 ml |

Adjust pH to 8.8 with 6N Hcl. Make to 150 ml with deionized water. Store at 4oC

0.5 M Tris-HCl, pH = 6.8:

|Tris Base/deionized water |6.0 g/~60 ml |

Adjust pH to 6.8 with 6N Hcl. Make to 100 ml with deionized water. Store at 4oC

5X Running Buffer, pH = 8.3 (dilute 70 ml with 280 ml to make lX):

|Tris base |9.0 g |

|glycine |43.2 g |

|sodium dodecyl sulfate (SDS) |3.0 % |

|deionized water |make to: 600 ml |

Store at 4oC. Warm to RT before use if precipitation occurs.

Fairbanks staining solution A

|Coomassie Blue |250 mg/250 ml DDH2O |

|Isopropyl alcohol |125 ml |

|Acetic Acid |50 ml |

|DDH2O |make to: 500 ml |

Fairbanks staining solution D

|Acetic Acid |50 ml |

|DDH2O |make to: 500 ml |

DATA SHEET

Team Members: Date: % Gel:

Lane

1 |Sample | |Lane

2 |Sample | |Lane

3 |Sample | |Lane

4 |Sample | |Lane

5 |Sample | | |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |

Lane

6 |Sample | |Lane

7 |Sample | |Lane

8 |Sample | |Lane

9 |Sample | |Lane

10 |Sample | | |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. |Dist |Rf |M.Wt. | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |

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