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pvdQ Expression Analysis Protocols

IPTG Induction

[PCR or Restriction Digestion Test must be performed to check for transformation of correct plasmid].

• Pick a colony and inoculate it into 5 mL broth with ampicillin. Incubate for 8 hours.

• Use 1mL of the culture in 10mL of broth with ampicillin (1:10 ratio). Grow the culture in warm room shaker till the OD reaches 0.6 (-0.8). Usually take around 3-6 hours.

• Add appropriate amount of IPTG (0.4-1.0 mM to the final concentration).

• Incubate at 37°C in shaker for 3-4 hours.

• Centrifuge and remove the supernatant (note the volume).

• Store the pellet in -20°C freezer until sonication.

Osmotic Shock to Obtain Periplasmic Fraction:

• Resuspend the pellet in 0.5mL PBS (phosphate buffered saline) or wash pellet twice in 30mM NaCl.
  [1L 1XPBS: 800mL Distilled Water, 8g NaCl, 0.2g KCl, 1.44g Na2HPO4-2H2O, 0.24g KH2PO4, adjust pH to 7.4 with HCl, Distilled water to a total of 1L. Isotonic and nontoxic to cells, so can be sued to dilute substances, wash reagent.]

• Centrifuge for 10min at 4°C (4863g). Discard the supernatant.

• Resuspend the pellet in 2.5mL ice-cold sucrose buffer.
  [Sucrose Buffer: 30mM Tris-HCl (pH 7.3), 1mM EDTA, and an equal volume of 0.5M sucrose.]

• Incubate for 10 minutes on ice while shaking vigorously.

• Centrifuge for 10min at 4°C (4863g). Discard the supernatant.

• Resuspend the pellet in 0.25 mL ice-cold 0.5mM MgCl2. Add appropriate amount of proteinase inhibitor.

• Incubate on ice for 10 minutes.

• Centrifuge for 20 minutes at 10,000g.

[Supernatant contains the periplasmic proteins. The pellet, which contains the rest of the cell components after the periplasmic components have been released into the supernatant, can be processed as the preparation of whole cell extracts. Then, the supernatant collected will contain the non-periplasmic cellular proteins.]

Sonication

• Add a volume equal to the volume of discarded supernatant of PBS to the pellet. Resuspend well.

• Add the proteinase cocktail mix (the volume added depends on its concentration and the volume of PBS).

• Place the micro centrifuge on ice. Elevate it to an appropriate height so as to properly submerge the sonicator's rod into the sample.

• Set the pulse (4 second), rest time (7 seconds), time (2 minutes), and amplitude (20%) to appropriate values. Place the sample on ice and elevate to an appropriate height.

• Turn on the sonicator and sonicate till the sample is clear (continually check the opacity at 45 second intervals).  

• Centrifuge at 13,200 rpm for 10 min. Remove save the supernatant and the pellet.

• Resuspend the pellet in equal volume of PBS (as the volume of the supernatant).

• Store in -20 degrees freezer until the samples are ready to use for SDS-PAGE/Western Blot.

SDS-PAGE

• Prepare 12% Seperating Gel corresponding to a 78kDa protein.

• Fill ¾ of the gel cassette with the Seperating Gel. Allow it to completely polymerize.

• Fill the rest of the cassette with the Stacking Gel. Let it polymerize for about one hour.

• Add 3X Protein loading buffer to 20uL of the protein samples. 5% (of the total volume of the dye) Mercaptoethanol should be added freshly. Mix, boil at 100 degrees for at least 5 min.

• Place the gel in the electrode assembly. Place the assembly into the tank.

• Fill with 1X Gel Tank buffer. Make sure interior of electrode assembly has equal or more buffer as outside.

• Attach to the power supply. Run at 110 V until the loading buffer reaches the bottom edge of the separating gel.

• Upon completion, disassemble by removing the gel from between the glass plates.
   

Western Blotting

• Perform the transfer

• Incubate the membrane with non-fat milk at room temperature for 30 minutes. This is the blocking step that prevents non-specific antibody binding.

• Place membrane in the primary antibody. Incubate at 16 degrees on roller overnight.

• Wash the membrane with Tris Buffer Saline and Tween20 (TBST) three times at 10-minute intervals. Place on shaker during the wash step.

• Incubate with secondary antibody on roller for one hour.

• Wash the membrane with TBST three times at 10 minute intervals.

• Place the membrane in an X-ray cover. Add the substrate, and immediately develop the X-ray film in a dark room.

Notes:
- Samples included IPTG induced and no IPTG samples for BL21 colonies with and without the recombinant plasmid. The BL21 without the plasmid serves as a control of the proteins already present within the bacterial strain. Also, for each sample, both 20uL of the pellet resuspended in PBS (containing insoluble proteins) and the supernatant (with soluble proteins and the expected pvdQ) were loaded in separate wells during electrophoresis. 
- Since the pvdQ protein contains a His Tag sequence at its C-terminal, the primary antibody used is Anti-His antibody derived from rabbits. The secondary antibody used is Anti-rabbit antibody derived from goat.

Croomassie Blue Staining

• Place the gel in a container and cover it with croomassie blue dye.

• Place in shaker for 20 min.

• Destain using ethanol/acetic acid/water solution. Destain at least 3 times in 10 min intervals on the shaker.

• Visualize the gel
   

His Tag Protein Purification

• Prepare a 200mL stock of 1X Charge Buffer, 1X Binding Buffer, 1X Wash Buffer, and 1X Elution Buffer.

• Column Preparation:
          - Add a few mL of ste1ile, ddH20 to the dry column. Gently apply pressure to the top of the column to allow the column flow to commence.
          - Mix the resin well and add 1 mL of the resin into the column. Allow it to settle.
          - Wash the column with 3 volumes of ddH20.
          - Wash the column with 5 volumes of 1X Charge Buffer.
          - Wash the column with 3 volumes of 1X Binding Buffer.

• Column chromatography:
          - Allow 1X Binding buffer to reach right at the surface of the column bed.
          - Load the column with the prepared protein extract.
          - Re-load the flow through to make sure all the HisTag protein binds to the resin.
          - Wash the column with 10 volumes of 1X Binding Buffer. This step can be repeated using the flow through.
          - Wash the column with 6 volumes of 1X Wash Buffer.
          - Elute the bound protein with 6 volumes of 1X Elution Buffer.
   

AHL Detection Assay

• Prepare 100ul sample of
      (a) IPTG induced whole cells    (b) Sonication supernatant    (c) Sonication pellet resuspenstion and their control by boiling them at at 100℃ for 15 minutes.

• Dissolve AHL in Acetonitrile organic solvent (final concentration of 0.5 mg/mL).

• Perform serial dilutions. Begin with 2ul of the acetonitrile C₁₂ - AHL stock and dilute to final concentrations of 10, 7.5, 5.0, 2.5, 1.25 and 0.625 ug/uL.

• Allow acetonitrile to evaporate

• Redissolve the C₁₂ - AHL in 100ul sample

• Duplicate Standards 100ul of C₁₂ - AHLs to final concentrations of 2500, 1250, 635, 312.5, 156.25, 78.125, 39.0625, 19.53125, 9.765625 mg/uL

• Incubate the tubes at 37℃, 70 r.p.m for 4 hours.

• 125uL of a 1:1 mixture of hydroxyl amine (2M): NaOH (3.5M) was aliquoted and mixed with the sample.

• 125uL of a 1:1 mixture of ferric chloride (10% in 4M HCl) : 95% ethanol was added and mixed well.

• Aliquot 150uL of each sample in a 96-well plate.

• Determine the amount of C₁₂ - AHLs in each sample spectrophotometrically at 520nm.