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Development (hemolymph)

Changes In Hemolymph During M. sexta Development

Protocol: SDS-PAGE Analysis of Manduca sexta Hemolymph Proteins

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Introduction

Gel electrophoresis has been used widely for the separation of proteins and nucleic acids. This technique involves the separation of the components of a sample within a matrix (gel) in the presence of an electrical field (electrophoresis). In this lab exercise, we will use polyacrylamide gel electrophoresis (PAGE) to separate the components of a complex protein mixture.

The polyacrylamide gel matrix is formed by the co-polymerization of two monomers: acrylamide and bis-acrylamide. The acrylamide polymerizes into long chains which are crosslinked at intervals by the bis-acrylamide, forming polyacrylamide. PAGE is especially useful because the gel and the buffer parameters can be chosen to provide separation of proteins by charge, size, or charge and size. For instance, the porosity of the matrix can be altered by changing the percentage of acrylamide used and/or the amount of crosslinker bis-acrylamide present. The higher the percentage of acrylamide in a gel, the more dense the gel and the better the separation of small proteins. In contrast, the lower the percentage of acrylamide, the more open the matrix which favors the separation of larger proteins.

In order to most accurately separate proteins according to their size, however, the proteins must be devoid of tertiary and quaternary structure (denatured). Dithiothreitol (DTT) has been included in the sample dye to reduce disulfide bonds. The anionic detergent sodium dodecyl sulfate (SDS) will be used as the denaturing agent in the gels and sample buffer. SDS is an amphipathic molecule with a long hydrophobic tail and a small negatively charged head group. The hydrophobic portion of the molecule coats the protein, disrupting the secondary, tertiary, and quarternary structure. Another consequence of treatment with SDS is that the proteins are artificially coated with negative charges. Therefore, in an electric field they migrate toward the positive end. Since the proteins are denatured (they have no distinct shape) and because the negative charge on the proteins is exceedingly large (so the charge differences between the proteins is negligible), the primary factor determining the rate of migration of the protein through the matrix is the size of the protein. The smaller proteins will migrate through the matrix fastest and will be closest to the 'bottom' of the gel.

 

Materials

per working lab group:

  • Pasteur pipettes and bulbs
  • bench diaper
  • micropipettors
  • micropipette tips for above
  • water (~ 50 ml)
  • 5 ml and 10 ml pipettes
  • pipette bulb/pump
  • filter paper squares
  • lab tissues
  • 50 ml side-arm flasks
  • ice bucket
  • foam sponge (optional)
  • 0.5 µl microfuge tubes
  • 5X SDS-PAGE sample dye (see below)
  • 10 µ l of molecular weight markers (e.g. BioRad or Sigma)

per pair of working lab groups:

  • squirt bottle of water
  • power supply
  • gel and electrophoresis apparatus (e.g. Mini-PROTEAN II Electrophoresis Cell from BioRad)

per class:

  • vacuum apparatus (in hood)
  • microfuge
  • rotary shaker
  • saran wrap
  • containers for staining gels

SOLUTIONS/BUFFERS

  • 10% ammonium persulfate solution (available from Sigma or BioRad)
  • TEMED (available from Sigma or BioRad)
  • 1.5 M Tris-HCl, pH 8.8 (Buffer B)
  • 10% SDS
  • 0.5M Tris-HCl, pH 6.8 (Buffer C)
  • Acrylamide (30%) stock (37.5:1) (available as pre-weighed from BioRad)
  • 5X Running buffer (0.12 M Tris-base, 0.96 M glycine, (pH 8.3) 0.5% SDS)
  • Coomassie Blue Stain (0.1% Coomassie blue R-250 in 40% MeOH, 10% HOAc)
  • Destaining Solution (40% methaonl, 10% acetic acid)
  • Destain II (5% methanol, 10% acetic acid)
  • 5X SDS-PAGE sample dye (15% beta mercaptoethanol, 15% SDS, 1.5% bromophenol blue, 50% glycerol)

 

Hazards:

Acrylamide has been shown to be a neurotoxin. Repeated skin contact, inhalation, or swallowing may cause nervous system disorders. ALWAYS wear gloves, safety glasses, and a lab coat when handling unpolymerized acrylamide or pouring gels. Rinse out your acrylamide pipettes immediately with lots of water. After you have poured your gel, leave the remaining solution in the flask to polymerize. After the lab period, scrape out the polymerized material onto your bench diaper and throw it away. Rinse the flask with water. If you do get acrylamide on your skin be sure to wash with soap and rinse thoroughly with water. Polymerized acrylamide is not harmful. TEMED is a flammable substance, and the vapors are irritating to mucous membranes and eyes. Contact with skin may cause irritation. Acetic acid is an irritant by skin contact or inhalation. Ammonium persulfate is harmful if swallowed, inhaled or absorbed through the skin. It is extremely destructive to mucous membranes. The hazards of methanol and SDS have previously been discussed.

Always turn the power supply off before removing the lid of the gel apparatus or working with it in any way. Failing to turn off the power supply can result in !!!!!!DEATH BY ELECTROCUTION!!!!!!

WEAR GLOVES, PROTECTIVE EYE WEAR, AND LAB COATS THROUGHOUT THIS LAB.

 

Method:

For each working group:

1. There are two components to the gel we will be using: a separating gel on bottom and a stacking gel on top.

Separating gel

10%

Distilled water

4.2 ml

1.5M Tris-HCl<pH 8.8

2.5 ml

10% SDS

100 µl

Acrylamide-bis (30% stock)*

3.3 ml

These materials may be mixed in the side-arm flask. Degas the solution by attaching it to a vacuum apparatus until bubbles stop forming at the surface (1-2 minutes).

Add:

10% ammonium persulfate

50 µl

TEMED

5 µl

Pour or pipette between the glass plates. Layer enough water over the surface of the acrylamide to form about a 2 mm layer.

 

2. When the separating gel has fully polymerized (20-40 minutes), carefully remove the water overlay by absorbing it with a piece of filter paper slid down between the two glass plates.

3. Allow any remaining acrylamide to polymerize in the flask and then scrap it out into a waste container in the hood. Rinse out the flask.

4. Prepare the stacking gel solution by combining the first four reagents in a 50 ml side-arm flask:

Stacking gel

4.0%

Distilled water

6.1 ml

0.5M Tris-HCl, pH 6.8

2.5 ml

10% SDS

100 µl

Acrylamide-bis (30% stock)*

1.3 ml

5. Degas the solution for 2-3 min. or until there are no bubbles present using the vacuum pump.

6. Add 50 µl 10% ammonium persulfate and 10 µl TEMED to the flask and swirl. Be sure to mix thoroughly by gentle (NOT vigorous) swirling. Be careful about volumes because the wrong volumes of catalysts will not allow the gel to polymerize.

7. Immediately pour the stacking gel using a Pasteur pipette. Pour down the side of the gel apparatus near one of the spacers. Pour slowly and be careful to avoid the formation of bubbles. Place the comb at a slant between the two glass plates and lower gradually. This helps to push air bubbles out the top.

8. Allow the gel to polymerize for 20-40 min.

9. While the gel is polymerizing, prepare your hemolymph samples for loading. In a 0.5 ml microfuge tube add 1.5 µl of hemolymph, 6.5 µl water, and 2µl 5X SDS-PAGE sample dye. If a determination has been made of the protein concentration, use a volume of hemolymph containing 8 ug of protein plus 2 ul of 5X SDS-PAGE sample dye and use water to bring the final volume to 10 ul.

10. You will also be loading a molecular weight marker on each gel so prepare the marker. Pipette 10 µl of the marker solution into a 0.5 ml microfuge tube. Quick spin all the samples in the microfuge to mix the samples.

11. See step 14. Make the diluted running buffer.

12. Heat your samples and molecular weight markers at 100ūC in the water bath for 3 minutes just before loading. Do not store the samples on ice after heating.

13. After polymerization of the gel is complete, take the casting stand to the sink and rinse with water to remove any non-polymerized acrylamide. Then remove the comb by pulling it straight up, slowly and gently. Rinse the wells with distilled water at the sink using a water squirt bottle.

14. Remove the gel assembly from the casting stand and snap it into the cooling core (see instructions on the Mini-PROTEAN II Cell Assembly Guide at your table). Assemble so that two gels are in one unit.

15. Lower the cooling core into the lower buffer chamber. Dilute the running buffer from 5X to make 300 ml of 1X buffer. Add a small amount of 1X running buffer to the top chamber. Check for any leaks. If no leaks are found, pour ~115 ml of 1X running buffer into the top chamber.

16. Pour the remainder of the running buffer into the lower buffer chamber so at least the bottom 1cm of the gel is covered.

17. Briefly microfuge all of the sample tubes (again, be sure to balance!) for 4-5 sec. to collect the contents.

18. Load your entire sample into the well slowly and carefully using one of the special pipette tips provided by the faculty member. Hints:

  • do not use the outside lanes on either side of the gel
  • load the gel in an asymmetric fashion (e.g. markers at one end, not in the middle)
  • write down the order in which the samples were loaded
  • don't forget to load the molecular weight markers

19. Place the lid on top of the lower buffer chamber. Attach the electrical leads to the gel apparatus. Be sure to match red to red and black to black.

20. Attach the leads to the power supply and turn on power. Set the power at 200 volts; the current should be approximately 60 mA per gel.

20. Run the gel for approximately 45 min. or until the dye just begins to run out the bottom.

21. Turn the voltage to zero and then turn off the power supply. Disconnect the electrical leads.

22. Remove the cell lid and pull inner cooling core out of the lower chamber. Pour off the upper buffer into the sink and flush with running water.

23. Disassemble the units to obtain your glass plates holding the gel.

24. Push one of the spacers of the glass sandwich out to the side of the plates and twist to remove the top glass plate.

25. Carefully rip off the stacking gel and gently remove the separating gel from the second glass plate by grasping two corners and lifting off. These gels are very thin and may rip easily. Be careful! This is easier to do if you have wet fingers. Nick one edge of the separating gel for orientation. Record in your notebook which edge you marked.

26. Place the gel in enough Coomassie Blue stain to cover. Stain the gel for at least 30 min. More than one gel can be stained in the same container if there is a way to distinguish between the gels (such as a nick in the corner).

27. Clean all glass, spacers, combs, and the apparatus thoroughly with soap and water.

28. Transfer the gel from the stain to the destain solution. Cover the container with saran wrap. Pour the Coomassie Blue back into its original bottle using the funnel. Leave the gel in the destaining solution on the shaker for 30 min.-3 hr. with a sponge to help soak up the dye. Do not leave overnight. After destaining, transfer the gels to destain II. Pour the destain back into the "used destain" bottle for recycling or disposal.

29. Gels can be stored in a ziplock bag or they may be dried.

 

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March 1999