Analysis of Proteins

Posted on by Leakey

Learning Goals
· Understand the concept of gel electrophoresis
· Electrophoretic separation of proteins
· Staining to visualize the proteins in the gel
· Gel dock imaging of stained gel
· Analysis of gel image and identification of protein bands
Principle
Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis is a protein analysis technique developed by Ulrich K. Laemmli to separate proteins based on their molecular weights. SDS is used in this method to linearize the native structure of protein and to provide an overall negative charge to the proteins. The polyacrylamide gel serves as the matrix which allows protein migration under the influence of electric current. The migration of proteins through the gel causes the separation of proteins by their molecular weights.

Figure 1: SDS-PAGE schematic

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Staining the gel
At the end of electrophoresis, the gel is removed from the cast and stained with a dye to detect the migrated proteins. This allows visualization of the separated proteins.

Figure 2: Staining with different dyes left- Coomassie blue, right- silver nitrate
Image analysis – Protein Ladder
The stained gel is imaged and protein bands are analyzed with reference to the protein ladder The protein ladder is a mixture of proteins of known molecular weights and is used in SDS-PAGE for identification of protein by estimation of their molecular weights.

Figure 3: Precision Plus Protein Dual Color Standard
Materials:
· XCell SureLock – 1
· Novex Tis-Glycine 10% Gel – 1
· Bacterial cell lysates and media supernatant – from previous lab (strains used: Escherichia coli, Serratia marcescens, Rhodococcus opacus)
· Protein Ladder – 10ul
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· Bovine Serum Albumin (BSA) standard – 25ul of 2.0mg/ml
· Power Pack- 1
· Staining and de-staining reagents (A-D)
· Box – 1
· Measuring Cylinder -2
· Styrofoam tub-1 (for ice)
· Timer – 1
· Sharpie – 1
· Tube rack -1

Procedure:
· Turn on the heat block, fill the wells with water, and set the temperature @70oC
· Get ice in the Styrofoam tube and gather the samples
· Dilute the samples in 2x sample buffer (total loading volume is 20 ul for the samples except protein ladder)
· Incubate the samples on the heating block for 10 mins.
· Place the ladder at room temperature to warm it.
· Prepare 350 ml of 1x running buffer using the stock 10x Tris-Glycine running buffer.
· Remove the tank from the box and start tank setup along with gel preparation simultaneously.

· Gel Preparation

  1. Cut open the gel cassette pouch with scissors and remove cassette.
  2. Drain away the gel packaging buffer.
  3. Remove the gel cassette from the pouch and rinse with deionized water.
  4. Note: Always handle the cassette by its edges only.
    · Tank setup
  5. Peel off the tape covering the slot on the back of the gel cassette.

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  1. Pull the comb out of the cassette in one fluid motion to expose the gel loading wells.
  2. Use a pipette to gently wash the cassette wells with 1X running buffer. Invert the gel and shake to remove buffer. Repeat twice. Fill the sample wells with running buffer. Note: Be sure to displace all air bubbles from the cassette wells as they will affect sample running.
  3. Lower the Buffer Core into the Lower Buffer Chamber so that the negative electrode fits into the opening in the gold plate on the Lower Buffer Chamber as shown in the figure.
  4. Insert the Gel Tension Wedge into the XCell SureLock® behind the buffer core. Make sure the Gel Tension Wedge is in its unlocked position, allowing the wedge to slip easily into the XCell SureLock® unit. The Gel Tension Wedge should rest on the bottom of the Lower Buffer Chamber.
  5. Insert gel cassettes into the lower buffer chamber.
  6. Place one cassette behind the core and the Buffer Dam in front of the core. The shorter “well” side of the cassette faces in towards the buffer core. The slot on the back of the cassette must face out towards the lower buffer chamber (see figure below).
  7. Pull forward on the Gel Tension Lever in a direction towards the front of the XCell SureLock unit until lever comes to a firm stop and the gels or gel/buffer dam appear snug against the buffer core (see figure below).
  8. When fully assembled, cassettes and Buffer Core are in place and Gel Tension Wedge is locked into position

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  1. The Upper Buffer Chamber (cathode) is the void formed between the cassette and the buffer dam on each side of the buffer core.
  2. Fill the Upper Buffer Chamber with ~200 mL of the appropriate running buffer (see page 18). Use enough running buffer to completely cover the sample wells.
  3. Ensure that the Upper Buffer Chamber is not leaking. If the level of running buffer drops, the electrophoresis core and cassettes are not properly seated. Repeat the tank setup.
  4. Fill the Lower Buffer Chamber with the rest of the buffer.

· Loading Samples

  1. Loading order left to right (1. Protein ladder, 2. BSA, 3. Media Supernatant A, 4. Corresponding Bacterial cell lysate A, 5. Blank, 6. Media Supernatant B, 7. Corresponding Bacterial cell lysate B, 8. Media Supernatant C, 9. Corresponding Bacterial cell lysate C, 10. Sample of choice.)
  2. Remove the samples from the heating block and allow it to cool for a minute.
  3. Spin the samples for a min to collect the evaporated sample
  4. Load 7 ul of the protein ladder and ~20 of each sample to the corresponding wells (note: lane order)

· Electrophoresis run

  1. Set the power pack at 225V constant mode and connect the lid wires to 250V output slot (color match the wires)
  2. Start the run and run till the samples migrate around half the gel length
  3. Stop the run and reduce the voltage to 125V constant mode and run till the samples migrate almost ¾ the length.
  4. End electrophoresis and disconnect the wires.

· Removing the gel from the cassette

  1. At the end of the run, turn off the power and disconnect the cables from the power supply.
  2. Remove the lid and unlock the Gel Tension Lever. There is no need to remove the Gel Tension Wedge.
  3. Remove the gel cassettes from the mini-cell. Handle gel cassettes by their edges only.
  4. Lay the gel cassettes (well side up) on a flat surface, such as the benchtop. Allow one edge to hang ~1 cm over the side of the benchtop.
  5. Carefully insert the Gel Knife’s beveled edge into the narrow gap between the two plates of the cassette.
  6. Note: Do not push the knife forcefully between the cassette plates or you may cut into the gel.
  7. Push up and down gently on the knife’s handle to separate the plates. You will hear a cracking sound which means you have broken the bonds which hold the plates together. Repeat until you have broken the bonds on one side.
  8. Rotate the cassette and repeat Steps 5–6 of this procedure until the two plates are completely separated.
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  9. Upon opening the cassette, the gel may adhere to either side. Remove and discard the plate without the gel, allowing the gel to remain on the other plate.
  10. Use the gel knife to cut the foot and well of the gel using gel knife
  11. Scoop and transfer the gel to the box using the gel knife. (if there is any difficulty pipette dI water between the gel and plate)
  12. Note: Be gentle to prevent ripping of the gel

· Staining and de-staining the gel

  1. After electrophoresis is complete, place the gel in a microwave-safe plastic container containing enough reagent A to cover the gel. (use ~50 mL of reagent A)
  2. Heat the gel in a microwave oven on full power until the solution is boiling (~2 min).
  3. Cool the gel for 5 min at room temperature with gentle shaking.
  4. Discard the used reagent A and rinse the gel briefly with H2O.
  5. Add ~50 mL of reagent B and heat the gel in a microwave oven on full power until the solution is boiling (~80 sec).
  6. Discard the hot reagent B and rinse the gel with H2O.
  7. Add ~50 mL of reagent C and heat the gel in a microwave oven on full power until the solution reaches the boiling point (~80 sec).
  8. Discard the hot reagent C and rinse the gel with H2O.
  9. Add ~100 mL of reagent D and heat the gel in a microwave oven on full power until the solution reaches the boiling point (~80 sec).
  10. Place a piece of laboratory tissue in the solution to absorb excess dye.
  11. Cool the gel for 5 min at RT on the shaker.
  12. Repeat Steps 9-11 twice more, or shake the gel in reagent D for 15 min or more at room temperature. (Until the gel is clear and the bands are visible)
  13. Note: always have enough reagent before boiling to prevent the gel from ripping.

· Imaging on Gel Dock

  1. Start computer and select the Image lab software
  2. In the protocol, select proteins and Coomassie blue, optimize for faint bands
  3. Wipe the tray and place the gel on the tray using the gel knife
  4. Insert the tray and run protocol.
  5. Save the image and export it to publishing format. Collect the data in a storage device.

Post-Lab Observations
Ø Was the lab successful? Explain
Ø If you were to repeat this lab what would you do to obtain better results?
Post-Lab Assignment
v Can gel electrophoresis be applied to nucleic acids? Explain and give examples of gel matrix used.
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v Does varying the concentration of polyacrylamide in the gel affect protein migration? Yes or no and explain.
v You run an SDS-PAGE with the following mixture of proteins: casein, ovalbumin, and BSA. List their order of migration from farthest to closest to the top of the gel.
v No protocol this week. Instead provide detailed explanation of the results you see on the gel image. Also, try to identify at least one protein based on its molecular weight (use protein data banks to find the protein name; search using species name and molecular weight)

Comments from Customer
Here are the instructions for post-lab question 4.

Go to NCBI website and select proteins in the database. Then perform a search using the name of the bacteria in the search box. (this will yeild a list of milions of protein for the corresponding bacteria)
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From the search results filter the protein using molecular weight. Estimate the molecular weight of the band you see on the gel and use a ± 2kDa as the range to filter. (conver kilo Daltons to Dalton before entering the custom range)

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Then select a protein and open the FASTA format of the protein from the hyperlink below the protein name

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Copy the FASTA format amino acid sequence of the protein

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Search for a protein molecular weight calculator. I use ExPASy but you can use any of the calculators.
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Paste the sequence in the text box and compute the molecular weight. (This step is to ensure that your protein molecular weight matches the molecular weight of the protein band on the gel)
An Ink Drawing

Since I have used E. coli, you guys try the other two strains.

The loading order of the lanes is as follows
Protein Ladder
BSA
R. opacus media supernatant
R. opacus cell lysate
Blank
S. marcescens media supernatant
S. marcescens cell lysate
E. coli media supernatant
E. coli cell lysate
R. opacus whole cells

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