ECL analysis of cyt c expressing strains

Whole Cell Lysis for Quantitative Analysis of Cytochromes in E. coli, Tris-HCl gel for Separation of Proteins and Quantitative ECL Staining of C-type Cytochromes
Authors: Heather Jensen, Matt Hepler, Cheryl Goldbeck, Caroline Ajo-Franklin
Goal: To lyse E. coli cells in such a way that specific cytochrome content per cell can be analyzed by ECL

Overview:

• In this procedure, E. coli cells are lysed by addition of lysozyme, which degrades the cell wall, and B-PER, a detergent which breaks open the cells.
• This procedure has two specific objectives that set it apart from more typical procedures to lyse whole cells. The first goal is to avoid conditions which would prevent detection of cytochrome c, i.e. beta-mercaptoethanol. The second goal is to permit quantitative comparisons of protein abundance by Western blotting or ECL detection of cytochromes. This requires that the lysate be amenable to (quantitative) pipetting and that we can determine the total protein content by the BCA protein quantification kit. Thus we have included DNAaseI to reduce the viscosity of the cell lysate (viscosity is mainly due to the presence of chromosomes). Additionally, we have minimized the mass of enzymes added to the cells so that it is small compared to the total protein from the cells, and added the serine protease inhibitor PMSF. The former aspect will permit quantitative measurement of the total protein per sample, so that each sample can be normalized to the total protein. The latter aspect should prevent degradation of proteins of interest. The concentration of cells has been diluted 3x relative to the standard protocol to obtain a cytochrome concentration convenient for diluting cells for loading a certain heme loading amount.

Critical Steps:
1. If you change number of cells, re-scale the protocol for a new [cell].

2. Use fresh DNAase I and lysozyme from powder.

3.Sodium Dithionite should be stored in a 4 oC fridge under Nitrogen.

Important Considerations:

The concentration of cells is less than in the protocol Whole Cell Lysis for Quantitative Analysis of Proteins in E. coli, because the concentration of cells in this protocol is more convenient for calculating the necessary dilution.  It is less than the concentration in Whole Cell Lysis for Quantitative Analysis of Proteins in E. coli, so the cells should be at least, but likely more, well lysed than in that protocol.

Materials:

• 20 mL of dense cell culture
• B-PER, Pierce #78248
• 50 mM EDTA
• 100mM MgSO4
• Chicken egg white lysozyme, catalog #
• 10mM HEPES pH7.4
• PMSF, catalog #
• Isopropanol
• DNAaseI, Sigma catalog #D5025
• 5M NaCl
• 4x LDS dye
• 4-20% Tris-HCl gel
• 1x TGS buffer
• Invitrogen Secure-Seal spacers, Invitrogen catalog #
• Sodium Dithionite
• Post-transfer nitrocellulose
• 15 mL Falcon tube or 13 mL snap top tube.
• 2 Sheet Protectors
• Biorad Blotting Transfer Apparatus (2 pads, gel box and holder)
• Pierce Transfer Buffer
• Chromatography paper
• 1 Cold pack 
• 5 mL Pierce Pico West Luminol/Enhancer
• 5 mL Pierce Pico West Stable Peroxide Buffer
• Small ziplock bag
• Porceau S stain
• Simply Blue SafeStain
• Imperial Blue (Pierce)
• 50% MeOH, 10% acetic acid

Procedure:

1. Transfer 2 aliquots of 10mL of cell culture into 2 15mL snap-top tubes. Label 1 aliquot “Diffuse Reflectance” and the second “Lysate”.

2. Normalize samples by weight using the scale in 5204 next to the PCR thermocyclers so that both weight 10g (assuming density 1g/mL). Tare the tubes before adding cell samples.

3. Pellet the cells by centrifuging for 10 minutes at 4000 rcf at 4C. This should correspond to ~20 mg of wet cell weight.

4. Resuspend the pellet of the Lysate sample in 2mL of B-PER.

5. To optimize function of lysozyme, add EDTA to a final concentration of 1mM

   1. (50mM)(v) = (2195uL total vol)(1mM) -> v = 44uL EDTA

6. For optimal function of DNAase, add Mg2+ to final concentration 4.2mM

   1. (100mM)(v) = (2195uL total vol)(4.2mM) -> v = 92uL MgSO4

7. Add chicken egg white lysozyme (Sigma) to final concentration of 0.012ug/uL. Make fresh each time.

   1. Make a 10ug/uL stock, and dilute it to 2ug/uL by adding 20uL of the stock solution to 80uL HEPES buffer pH 7.4.

   2. (2ug/uL)(v) = (0.012ug/uL)(2195uL) -> v = 13uL lysozyme stock

   3. Important note: Do NOT use lysozyme cocktails. These commonly include reducing agents.

8. Make up fresh stock DNAse I to a concentration 2U/uL (or 1 mg/mL) in 0.15M NaCl. Add stock solution to lysate sample such that its final concentration is 0.02U/uL. Make fresh each time.

   1. Add 60uL 5M NaCl to 1940uL ddH20 to make 0.15M NaCl.

   2. Dissolve 10mg DNAaseI in 1mL 0.15M NaCl.

   3. Make a 1:10 dilution by transferring 100uL of 10mg/mL DNAaseI into 900uL of 0.15M NaCl to get 2U/uL DNAaseI.

   4. (2U/uL)(v) = (2195uL total volume)(0.02U/uL) -> v = 22uL 1mg/mL DNAaseI stock

9. Add PMSF to a final concentration of 1mM. Make fresh each time.

   1. Make a 100mM stock fresh in isopropanol so that the final concentration is17.4mg/mL.

   2. (2195uL)(1mM) = (100mM)(v) -> v =22uL PMSF stock

10. Incubate Lysate sample at RT for 60 minutes.

11. As the cells are lysing, resuspend the Diffuse Reflectance sample in 10mL of 10mM HEPES pH 7.4.

12. Make a 1/10 dilution of these cells by transferring 15uL of cells to 135uL of 10mM HEPES pH 7.4.

13. Take OD600 of the 1/10 dilution on the UV-VIS in 5204, and calculate the OD600 of the undiluted cells by multiplying the resulting OD600 by 10.

14. If the OD600 are not between 3-4, concentrate or dilute the cells as necessary to get an OD600 between 3-4.

15. Make fresh 1M Sodium Dithionite.

16. Add 5uL 1M sodium dithionite to the 500uL cell suspension to give a final concentration of 10mM.

17. Transfer 400uL of the reduced cell suspension to an assembled Invitrogen Secure-Seal Diffuse Reflection cuvette.

18. Take the Diffuse Reflectance spectrum. (See Measuring Cyt c content of intact bacterial cells using diffuse reflectance.)

Calculate the background corrected A552nm using the formula:

A552nm,norm = (A552nm – 1.04*A570nm)

Determine the heme c concentration of the Diffuse Reflectance sample by applying:

[heme c] = A552nm,corrected/(ε*b’),

here ε has been experimentally determined for the diffuse reflectance cuvettes to be 0.08cm, and b’ is the per heme extinction coefficient for the cytochrome you are using.  For MtrC and MtrA, b’ is 28,000M-1cm-1.

19. The Lysate sample is 5x more concentrated than the Diffuse Reflectance sample, multiply the [heme c] calculated in step h. by 5 to determine the [heme c] concentration of the Lysate sample.

20. Calculate and prepare the dilution of the Lysate sample, into 1mL total, necessary to obtain a [heme c] concentration of 0.72uM, label this stock Loading Sample.  This concentration will allow for a loading of 16pmoles of [heme c] from this stock.

21. Prepare a dilution series from the Loading Sample according to the following table. Pipette slowly to avoid bubbles.

 

 

Running Gel

1. Transfer 60uL of each dilution into a fresh Eppendorf tube.

2. Add 20uL of 4x LDS dye to 60uL of each sample

3. Heat samples at 95 °C for 5 min in water bath.

4. Remove comb from gel and rinse out wells with Millipore water.

5. Load the gel into the gel box. Gel should have numbers facing out of the gel box. Fill the tip of the gel to the brim and the gel box to the fill line with 1x TGS buffer.

6. Load 30uL into your 4-20% Tris-HCl gel. Also load 15uL ladder into each side of the gel.

7. Run gel at 200 V for 60 minutes. Run until pink portion of the LDS dye runs off the gel.

8. While gel is running, find the spatula. Also, make 2L Pierce Western Transfer buffer from 10x stock and cold Millipore water 10min before the gel finishes running. Keep buffer on ice.

9. Break the gel container using the spatula to twist and starting from a bottom corner of the container. Gel is fragile! Use a pipette tip to scarp along edge of gel before attempting to remove.

10. Rinse gel twice with approximately 25mL Millipore Water.

11. Cut off bulge at bottom of gel and wells at top using the spatula. Also, cut off the bottom of the gel that doesn’t lie flat and a corner so you remember which side of the gel faces up.

12. Incubate gel 15 minutes in cold Pierce Western Transfer Buffer on shaker set to about 45. Make sure the gel is facing up.

13. While gel is incubating, pre-wet nitrocellulose.

    1. Cut chromatography paper into the same size as the pads.
    2. Open gel holder in BioRad Transfer Apparatus by putting the black side in the apparatus and the red side propped up against the slant of the apparatus.
    3. Put a pad on the black side, then a chromatography paper.
    4. Put a pad on the other side of the apparatus, then a chromatography paper, than the nitrocellulose (try to only touch sides of nitrocellulose).
    5. Pour enough Pierce Transfer Buffer into apparatus to cover everything.
    6. Load gel straight onto Biorad Transfer Apparatus on top of the chromatography paper. Do not flip gel while transferring.
    7. Place nitrocellulose on the gel, and press out bubbles with the spatula. Follow with the 2nd sheet of chromatography paper and the 2nd pad. Make sure everything is lined up. Nitrocellulose should be closer to the red side.
    8. Close the holder and load the holder into a half-full transfer box (filled with Pierce Transfer Buffer) with a cold pack and stir bar in place.  The red side of the holder should face the red side of the transfer box.
    9. Fill the rest of the transfer box with transfer buffer until the line.
    10. Transfer at 30V for 100 min while with the stir bar set to high speed. Check periodically to ensure that the mAmp is about 200.
    11. To clean up the transfer apparatus and everything else, rinse with water until there are no more buffers.

ECL staining and vizualization

14. Place nitrocellulose face up in gel tray. Rinse nitrocellulose with Millipore water immediately after transfer.

15. Cut off the same lower corner on the nitrocellulose as the gel such that when both are facing up, the cut corner of the nitrocellulose is on a different side than the cut corner on the gel (eg. Left instead of right).

16. Visualize transfer by Ponceau S stain.

    1. In the BioRad gel container, add enough Ponceau S to cover sheet, mix gently for a few seconds.

    2. Transfer Ponceau S back to storage container.

    3. Rinse sheet with Millipore water, should be able to see protein bands clearly.

    4. Destain by rinsing several times in Millipore water until almost no bands are visible.

17. Store nitrocellulose in Millipore water.

18. Mix 5mL Pierce Pico West Luminol/Enhancer with 5 mL Pierce Pico West Stable Peroxide Buffer in a 15 mL Falcon tube, just before use. Take care to not contaminate the stock bottles with the other solution. This mixture must be disposed of in the waste stream.

19. Move nitrocellulose to a small plastic bag after allowing excess water to drip off sheet.

20. Transfer Pico West Luminol/Enhancer solution to plastic bag and lay bag horizontally. Press air bubbles to opening of bag so nitrocellulose sheet is completely covered by Luminol/Enhancer solution.

21. Incubate nitrocellulose in Pierce ECL Substarte for 5 min, setting gel rocker to ~45 and ensuring that the substrate is covering the nitrocellulose.

22. Remove the sheet. Collect excess liquid by holding the sheet lengthwise perpendicular to the floor and touching the bottom corner with a Kim Wipe.  Place the nitrocellulose sheet in a sheet protector so it does not dry out.

23. Place the sheet protector on the appropriate loading tray so that the side of the nitrocellulose on which the proteins were transferred is facing up. Place the tray in the Gel Imager. Select Chemiluminescence and set exposure times. Try 1 min, 3 min, and 6 min for a total of 10 min.

24. Store nitrocellulose in Millipore water on the shaker in preparation for protein staining.

Staining the Gel for Total Protein

25. Rinse gel with about 100mL Millipore water 3 times for 5min to remove SDS and buffer salts, which interfere with binding of the dye to the protein.

26. Add enough SimplyBlue SafeStain to cover (about 20mL).

27. Shake at about 45 for 1hr at room temperature.

28. Discard the stain in the waste stream and wash the gel with water a few times (washes can go down the drain).

29. Let shake for another hour at room temperature in 100mL Millipore water. (Optional) Increase the sensitivity by adding 20 (30) mL 20% NaCl (w/v).

30. To obtain the clearest background for photography, perform a second 1-hour wash with 100mL water. Note: Sensitivity will now decrease if the gel is allowed to stay in the water for more than 1 day.

31. Pour out the water and put the gel in a sheet protector.

32. Place the gel on a white background so that it is face-up and load into the Chemidoc. Select Epi-light and set to auto-expose.

Stain the Nitrocellulose for Total Protein

33. Place nitrocellulose membrane containing transferred proteins in a tray and rinse for 1-2 minutes with Millipore water.

34. Mix the Imperial™ Protein Stain immediately before use by gently inverting or tipping/swirling the bottle several times.

35. Pour enough Imperial Protein Stain onto nitrocellulose to cover (about 10mL). Put on gel rocker for 2-5min. Gel rocker should be set to ~45.

36. Pour stain into waste stream. Keep nitrocellulose wet in Millipore water while you make destaining solution.

37. To make destaining solution, add 10% acetic acid and 50% methanol by volume to water.

• 1. Measure out 250mL methanol in fume hood. Pour excess methanol into waste stream for destaining solution.

  2. Mix methanol into 200mL water.

  3. Measure out ~50mL acetic acid in a beaker in fume hood. Pour into graduated cylinder to get exactly 50mL. Wear goggles and apron while doing this.

  4. Add acetic acid slowly to methanol and water.

  5. Pour excess acetic acid into waste stream.

  6. Let beaker and cylinder used to measure acetic acid dry before washing in sink.

45. Pour Millipore water down the sink and add enough destaining solution onto nitrocellulose to cover. Put on gel rocker for 4-10min. Gel rocker should be set to ~45.

46. Pour destaining solution into waste stream.

47. Pour new destaining solution onto nitrocellulose and let sit on gel rocker for another 4-10 min.

48. Repeat 2-3 times until you feel that no more stain is being pulled off. All destaining solution should go into waste stream.

49. Place the nitrocellulose in a sheet protector. Place the sheet protector on the white tray such that the nitrocellulose is facing up. Put the tray into the ChemiDoc.

50. Select Epi-light and set to Auto-expose.

51. Dump used destain into waste stream.

52. If store nitrocellulose in Millipore water after taking image, water can go down the drain.