Protocol x
 

• Type of paperLab Report
• SubjectNature
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Materials and Methods (15 marks) This should be a summary of the experimental procedures, written in your own words in the past tense and third person passive. You should always write in paragraphs and you must not use bullet points. Under no circumstances should you ever just reproduce the text in the Workshop manual.

Introduction

In

terms of total production tonnages used for food, wheat is currently second to rice as the main human food crop and is the leading source of vegetable protein in human nutrition (Nutrient Data Laboratory). Aphids (Order Hemiptera) are major insect pests of world agriculture, damaging crops by removing photoassimilates and vectoring numerous plant viruses (Smith and Boyko,

200

7

). The grain aphid (Sitobion avenae) is considered a serious pest of commercial wheat in the UK. Many aphid species can develop resistance to insecticides (Devonshire and Field, 1991), and restrictions on the availability of active ingredients for insecticide production in Europe (European Directives 91/

4

14/EEC) has prioritized research on crop varieties with resistance to aphid pests in UK agriculture (Painter, 19

5

1; Panda and Kush, 1995; Smith, 2005).

Most commercial wheat varieties have very little resistance to aphid pests (Lee, 1984; Dedryver and Di Pietro, 1984; Di Pietro and Dedryver, 1986; Migui, 2002; Migui and Lamb, 2003), with at best partial antibiosis, antixenosis and tolerance in some winter varieties (Lowe, 1984a; Lowe, 1984b; Havlícková, 1993). Wheat genetics are more complicated than that of most other crop species. Some wheat species are diploid, with two sets of chromosomes, but many are stable polyploids, with four sets of chromosomes (tetraploid) or six (hexaploid). Modern wheat varieties grown in the U.K. are hexaploid and have low genetic diversity for insect resistance traits (Ferry et al, 20

11

; Ogbonnaya et al, 20

13

; niab.com).

Diploid wheat (T. monococcum) lines have been observed to exhibit high levels of resistance against S. avenae (Migui and Lamb, 2003; 2004; Ferry et al, 2011). However, no previous studies have been carried out in these lines to investigate differentially expressed genes in response to aphid infestation. Therefore, differential proteomic analysis is being employed to identify putative defence responses in diploid wheat lines (Triticum monococcum L.) when subjected to grain aphid (S. avenae) feeding in order to better understand the basis of this observed resistance/tolerance.

In this lab you will conduct a 2D-electrophoresis separation of the wheat leaf proteome.

Methods

It is essential that you retrieve the full lab manual produced by GE healthcare for 2D electrophoresis:

Step 1 – Sample Solubilization

This has been done for you.

In this experiment 200mg of wheat leaf was ground in liquid nitrogen to a fine powder. The protein pellets available to you have been incubated in 10% (w/v) trichloroacetic acid/ acetone with 0.07% v/v 2-mercaptoethanol at -20 °C for 16 hours and then centrifuged at 35 000 x g for 20 mins.

> The pellets were washed with ice-cold acetone (0.07% 2-mercaptoethanol) 4-6 times and finally incubated at -20 °C for 1 h, and centrifuged at 12 000 x g for 15 min.

>>An acetone wash using 1 part sample: 3 parts acetone (w/v) was performed, the sample was vortexed to disperse pellet in acetone, and centrifuged at 13000 x g for 5 mins and this was repeated four to six times. This removes contaminating pigments. After the final wash the sample was transferred to the -20 °C freezer for 1 h, before retrieving and centrifuging.

You can read more about sample preparation for 2D-gel electrophoresis in Chapter 1 of 2-D Electrophoresis – Principles and

Methods

(GE Healthcare)

To solubilize the proteins, we use a conventional solubilization mixture that contains 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 20 mM DTT and 0.5 % IPG buffer. Urea and thiourea are used to unfold the proteins; DTT is added to reduce

Table 1

The volume range of the assay is 1–50 μl and the linear range for quantification is 0–50 μg. This corresponds well with typical volumes and concentrations of samples prepared for 2-D electrophoresis. 

Preliminary Preparation

Prior to performing the assay, prepare an appropriate volume of working colour reagent by mixing 100 parts of colour reagent A with 1 part colour reagent B. Each individual assay requires 1 ml of working colour reagent.

So how much will you need? Calculate now! Remember to count your sample!

Standard Procedure

1. Prepare a standard curve (in duplicate) according to the table below using the 2 mg/ml Bovine serum albumin (BSA) standard solution provided with the kit. Set up six tubes and add standard solution according to the table. Tube 1 is the assay blank, which contains no protein.

Tube number

1 2 3 4 5 6

Vol. of BSA St (μl) 0 5 10 15 20 25

Protein quantity (μg) 0 10 20 30 40 50

2. Prepare additional tubes containing 5, 10 or 50 μl of the sample to be assayed. Duplicates are recommended. The useful range of the assay is 0.5–50 μg and it is also recommended that more than one sample volume or dilution be assayed for each sample to ensure that the assay falls within this range.

3. Add 500 μl precipitant to each tube (including the standard curve tubes). Vortex briefly and incubate the tubes 2–3 min at room temperature.

4. Add 500 μl co-precipitant to each tube and mix briefly by vortexing or inversion.

5. Centrifuge the tubes at a minimum of 10 000 × g for 5 min. This sediments the protein.

6. Remove the tubes from the centrifuge as soon as centrifugation is complete. A small pellet should be visible. Decant the supernatants. Proceed rapidly to the next step to avoid resuspension or dispersion of the pellets.

7. Carefully reposition the tubes in the microcentrifuge as before, with the cap-hinge and pellet facing outward. Centrifuge the tubes again to bring any remaining liquid to the bottom of the tube. A brief pulse is sufficient. Use a micropipette to remove the remaining supernatant. There should be no visible liquid remaining in the tubes.

8. Add 100 μl of copper solution and 400 μl of distilled water to each tube. Vortex briefly to dissolve the precipitated protein.

9. Add 1 ml of working colour reagent to each tube (See “Preliminary preparations” for preparing the working colour reagent). Ensure instantaneous mixing by introducing the reagent as rapidly as possible. Mix by inversion.

10. Incubate at room temperature for 15–20 min.

11. Read the absorbance of each sample and standard at 480 nm using water as the reference. The absorbance should be read within 40 min of the addition of working colour reagent

Note:
Unlike most protein assays, the absorbance of the assay solution decreases with increasing protein concentration. Do not subtract the blank reading from the sample reading or use the assay blank as the reference.

12. Generate a standard curve by plotting the absorbance of the standards against the quantity of protein. Use this standard curve to determine the protein concentration of the samples.

For full protocol see:

https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314729545976/litdoc28954714AE_20110830215136

Lab 1b – Cleaning up samples using 2-D Clean-Up Kit

The 2-D Clean-Up Kit is designed to prepare samples that would otherwise produce poor 2-D results due to high conductivity, high levels of interfering substances, or low concentration of protein.

Current methods of protein precipitation suffer from several significant disadvantages:

· Precipitation can be incomplete, resulting in the loss of proteins from the sample and introduction of bias into the 2-D result.

· The precipitated protein can be difficult to resuspend and often cannot be fully recovered.

· The precipitation procedure can itself introduce ions that interfere with first-dimension IEF.

· Precipitation can be time-consuming, requiring overnight incubation of the sample.

A 2-D Clean-Up Kit circumvents these disadvantages by providing a method for selectively precipitating protein for 2-D electrophoresis. Protein can be quantitatively precipitated from a variety of sources without interference from detergents, chaotropes, and other common reagents used to solubilize protein. Recovery is generally greater than 90%. The procedure does not result in spot gain or loss, or changes in spot position relative to untreated samples. The precipitated proteins are easily resuspended in 2-D sample solution. The procedure can be completed in less than one hour.

The overall quality of protein separation using 2-D Clean-Up Kit has been shown to be superior to that of samples prepared by precipitation with acetone. Preparation of protein samples with the kit reduces horizontal streaking, improves spot resolution, and increases the number of spots detected compared with samples treated by other means

Protocol:

1. Transfer 1–100 μl of protein sample (containing 60 μg protein) into a 1.5-ml microcentrifuge tube (you need the quantification result to know volume required).

2. Add 300 μl of precipitant. Mix well by vortexing or inversion. Incubate the tube on ice (4–5 °C) for 15 min.

3. Add 300 μl of co-precipitant to the mixture of protein and precipitant. Mix by vortexing briefly.

4. Position the tubes in a microcentrifuge with cap-hinges facing outward. Centrifuge at maximum speed (at least 12 000 × g) for 5 min. Remove the tubes from the microcentrifuge as soon as centrifugation has finished.
A small pellet should be visible.

Proceed rapidly to the next step to avoid resuspension or dispersion of the pellet.

5. Remove as much of the supernatant as possible by decanting or careful pipetting. Do not disturb the pellet.

6. Carefully reposition the tubes in the microcentrifuge with the cap-hinges and pellets facing outward. Centrifuge the tubes briefly to bring any remaining liquid to the bottom of the tubes. Use a pipette to remove the remaining supernatant. There should be no visible liquid remaining in the tubes.

7. Without disturbing the pellet, layer 40 μl of co-precipitant on top of each pellet. Incubate the tubes on ice for 5 min.

8. Carefully reposition the tubes in the centrifuge with the cap-hinges facing outward. Centrifuge for 5 min. Use a pipette to remove the supernatant.

9. Pipette 25 μl of distilled or deionized water on top of each pellet. Vortex each tube for 5–10 s. The pellet should disperse, but not dissolve in the water.

10. Add 1 ml of wash buffer (prechilled for at least 1 h at -20 oC) and 5 μl of wash additive to each tube. Vortex until the pellets are fully dispersed.
Note: The protein pellet will not dissolve in the wash buffer.

11. Incubate the tubes at -20 °C for at least 30 min. Vortex for 20–30 s once every 10 min. At this stage, the tubes can be stored at -20 oC for up to one week with minimal protein degradation or modification.

12. Centrifuge the tubes at maximum speed (at least 12 000 × g) for 5 min.

13. Carefully remove and discard the supernatant. A white pellet should be visible. Allow the pellet to air dry briefly (no more than 5 min).

Do not over-dry the pellet. If it becomes too dry, it will be difficult to resuspend.

14. Resuspend each pellet in an appropriate volume of rehydration or IEF sample loading solution for first-dimension IEF (see Table 2). Vortex the tubes for at least 30 s. Incubate at room temperature. Vortex or aspirate and dispense using a pipette to fully dissolve.

15. Centrifuge the tubes at maximum speed (at least 12 000 × g) for 5 min to remove any insoluble material and to reduce any foam. The supernatant may be loaded directly onto first-dimension IEF or transferred to another tube and stored at

-80oC for later analysis.

Sample resuspension volumes

16. Table.2..Sample volumes for different Immobiline DryStrip gel lengths.

The optimal quantity of protein to load varies widely depending on factors such as sample complexity, the length and pH range of the Immobiline DryStrip gel, and the method of visualizing the 2-D gel separation.

Lab 1c – Loading the Sample

Six samples can be loaded at a time; it will take about 10 mins each to load your strip. Arrange with Dr Ferry and Nanda Puspita to do this as each set of samples run overnight and will be collected and frozen ready for lab 2.

Protocol:

Starting the IEF

Program 1

Passive rehydration 16:00 h at rt

Step 1 : 100 V for 0:40 h

Step 2 : 200 V for 0:20 h

step 3 : 450 V for 0:15 h

step 4 : 750 V for 0:15 h

step 5 : 2000 V for 0:10 h

step 6 : gradient for a total of 4000 Vh

The entire IEF takes approx. 24 h to finish. At the end of an IEF run strips will be stored at -80 C for lab 3.

You can read more about recommended IEF protocols for different IPG strip lengths and types in 2-D Electrophoresis – Principles and Methods (GE Healthcare).

Lab 2 – Second Dimension – SDS-PAGE

SDS-PAGE gels should be prepared as detailed in Appendix 1.

Equilibration of the Strips

The equilibration fulfils two functions: the disulfide bridges are reduced and the cysteine residues alkylated with iodoacetamide and the proteins are equilibrated with SDS so that they become negatively charged for the electrophoresis.

Prepare one plastic tube with lid that is filled with 10 ml fresh (or just thawed from the freezer) equilibration solution containing 65 mM DTT (add 100 mg DTT to 10 ml equilibration solution and invert the tube until it is dissolved) and one tube with 10 ml equilibration solution containing 135 mM iodoacetamide (250 mg iodoacetamide per 10 ml).

Equilibrate the IPG strips with gentle agitation first for 15 min in equilibration solution containing 65 mM DTT and then for 15 min in equilibration solution containing 135 mM iodoacetamide. Finally briefly rinse the strips with a few ml SDS-running buffer.

Risk Assessment and Precaution

Iodoacetamide is a strong alkylating agent and toxic. Please, protect yourself by wearing laboratory coats. Although iodoacetamide is toxic wearing gloves can be a disadvantage because of static electricity that can cause this chemical to spread over the bench. However, if you handle this chemical without gloves, be careful to avoid any skin contact and wash your hands afterwards. When you weigh up solid iodoacetamide, make sure that you do not contaminate the balance and the bench and expose other people to health risks. Spilled iodoacetmide can be wiped up with a humid paper tissue that then is sealed in a plastic bag. The group leader will take care of the final disposal.

Used equilibration solution that comprises iodoacetamide can be disposed of easily as follows. Dissolve excess solid cysteine in the equilibration solution to alkylate the iodoacetamide. Wait for about an hour until the bromophenolblue has turned from blue to green/yellow and discard the solution into the sink.

Cutting the Strips (optional)

You may find it easier to apply the equilibrated strips to the second dimension gel if approximately 1 cm plastic is cut off either the anodic or cathodic ends of the strips.

Loading a Strip onto the Polyacrylamide gel

SDS electrophoresis

The SDS electrophoresis is started at a constant current of 10 mA per gel for 1 h followed by a constant current of 40 mA per gel (maximum power 13 W/gel) for 4-5 h. After the SDS-electrophoresis the gels are fixed for at least one hour with 30% (v/v) ethanol in 10% (v/v) acetic acid.

Staining with Coomassie blue

Protein spots are stained with colloidal Coomassie blue G-250 (Sigma) and de-stained in water.

Scanned gels may be collected the following day.

Instructions and Marking Scheme for your Workshop write-up: GREEN BIOTECHNOLOGY 2017

Lab Portfolio

This will be written as a single document and will cover all the laboratory work that you do this semester.

1. Protein Quantitation (1 session)

2. Analysis of the plant proteome (1 session)

Workshop Report (portfolio)

Marking Scheme

Your report should contain:

1.
Title and Objectives of the workshop

(5 marks)

2.
Introduction

(25 marks)

The introduction should give the reader some background information on the subject in the report. You should carry out a brief literature review (1 – 2 pages) on analysis of plant proteomes and of wheat-aphid interactions and plant stress, adding appropriate references to your text. You should end the introduction by describing what you are going to do in the experiments.

3.
Materials and Methods

(15 marks)

This should be a summary of the experimental procedures, written in your own words in the past tense and third person passive. You should always write in paragraphs and you must not use bullet points. Under no circumstances should you ever just reproduce the text in the Workshop manual.

4.
Results and Discussion

(40 marks)

In the Results section you should describe the results you obtained.
Some examples
would include: did you recover protein from the extraction? Was the quantification accurate? Is your 2D gel clearly resolved? Answer these questions in sentences as part of your Results section and do not repeat the questions! The gel picture must be properly labelled on both the horizontal and vertical axes. In the Discussion, you should expand on the results you obtained, basing your discussion on the published literature.
Some examples
would include: Did the experiment work, how could it be improved? Did your gel look similar to published images of plant leaf proteomes? Did you recover all plant leaf proteins? How could you analyse the total leaf proteome? What are the uses of proteome profiling? You should make some informed suggestions, using the literature, about the results you have obtained. It does not matter if your comments are correct, just that you provide evidence that you have understood the theory behind the experimental study.

5.

Conclusion

(5 marks)

End the write-up with a statement summarizing the conclusions that may be drawn from the results obtained.

6.
References
(10 marks)

Compile a reference list using the Harvard system. References should also be quoted in the appropriate place in your text.

Marking criteria Lab Portfolio

5

4

2-3

0-1

Appendix 1

SDS-PAGE gel preparation

Second Dimension – SDS-PAGE

This is a continuous gel system and requires no stacking gel

1. Assemble 1x gel casting cassette and place it in the gel casting rig with the shaped glass plate towards the back making sure the plate is flush at the top and bottom of the rig before you tighten the plastic screws to secure them.

2. Now, place the assembled rig onto the base plate and while pressing down, turn the thumb screws to lock the two together forming a seal. Test the seal by pipetting approximately 4ml of distilled water into the cassette, if there is a leak, empty and repeat the procedure until you are confident there is no leak, then empty the water again.

3. Prepare the continuous resolving gel as indicated in Table 1 (p 3) and mix the solution by slowly pipetting up and down using a 1ml pipette,
Note:
Acrylamide is a neurotoxin so handle with care

4. Using the same pipette, load the solution into the gel cassette leaving a space of 5mm from the top of the glass back plate. Now, tap the rig slightly to remove any air bubbles.

5. Immediately overlay each gel with n-butanol (50/50 (v/v) with dH2O).

6. Leave the gel to polymerise (about 30 min- the remainder of the gel solution left in your beaker will serve as an indicator for setting). When the gel has polymerised, pour off the n-Butanol and rinse 3-4 times with ddH2O using a 1ml pipette.

7. Invert the gel rig and leave to drain off on some blue roll for a few minutes.

8. While the gel cassette is placed horizontally on the bench, use two small spatulae to place the cut IPG strip carefully on top of the polyacrylamide gel leaving enough room to load the small piece of filter paper which has been pre-soaked with the Marker proteins (Figures 1 & 2).

9. The strip is sealed with warm 0.5% (w/v) agarose dissolved in running buffer containing a trace of bromphenol blue.

10. Carefully, detach the gel rig from the base and insert the gel rig/cassette into the electrophoresis tank.

11. Fill the void between the two cassettes to the top with running buffer (0.025 mM Tris, 0.192 M glycine, 0.1% SDS, pH 8.3).

12. Place the lid onto the tank making sure the electrodes on the lid correspond to those on the rig.

13. Plug the electrode leads into the power source and run at 100V for approximately 1 – 1½ hours, or when the leading dye is nearing the bottom of your gel.

14. Switch the power off and disconnect the electrode leads, take the gel rig out of the tank and carefully loosen the screws to free your gel cassette.

15. Pour the InstantBlue stain solution into a small white tray and carefully dismantle the gel cassette. Place the gel into the stain and leave to incubate on a rocking platform set at slow speed for 5-15 minutes until protein spots become visible.

You should take a picture of the stained gel to put in your lab report.

Table 1. 12% Polyacrylamide Resolving Gel

Diagrams 1

Diagrams 2