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	==IgG protease activity assay==		==IgG protease activity assay==
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		+	Based on an experiment by [http://www.ncbi.nlm.nih.gov/pubmed/20441890 Abuknesha, ''et al'' (2010)].
	===Materials===		===Materials===

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Latest revision as of 09:20, 22 October 2010

Contents 1 Competent cells (Nina & Johan) 2 Competent cells (Andreas & Mimmi) 2.1 Materials 2.2 Procedures 3 Transformation (Nina & Johan) 4 Transformation (Andreas & Mimmi) 5 Quick transformation (Andreas & Mimmi) 6 Colony PCR verification (Andreas & Mimmi) 7 Site-directed mutagenesis 8 SDS-PAGE mixtures (Nina & Johan) 9 Mini prep(Nina & Johan) 10 Agarose gel clean up (Nina & Johan) 11 IgG protease activity assay 11.1 Materials 11.2 Procedures 11.2.1 Day 0 11.2.2 Day 1 11.2.3 Day 2

Competent cells (Nina & Johan)

From Morten Nørholm at the Department of Biochemistry & Biophysics Stockholm University

1. Add 5 ml LB in two 50 ml falcon tubes. Add the top of a tip bacteria into the two 5 ml LB. Grow ON in shake incubator 37 degree C.
2. Subculture each 5 ml of starterculture into two 400 ml pre-warmed LB. Grow at 37 degree C until OD reaches 0.6.
3. Put cells on ice for 20 min.
4. Harvest cells at 4000 rpm for 20 min, 4 degree C.
5. Discard supernatant if it looks clear (or spinn longer if it is cloudy).
6. Resuspend pellet carefully in 500 50 mM CaCl2 for a 1000 ml cell culture (1/2 the orifinal volume).
7. Put cells on ice 20 min.
8. Repeat step 5.
9. Resuspend cells in 16 ml CaCl2 + 15% Glycerol for a 800 ml starter culture (1:50 volume)
10. Put metal blocks in -80 degree C.
11. Snapfreeze 100 mikroL aliquots in ice-cold Epps (in pre-chilled blocks). Store in -80 degree C freezer.

Competent cells (Andreas & Mimmi)

Based and modified from the [http://openwetware.org/wiki/TOP10_chemically_competent_cells Top10 protocol by the Knight lab]

Materials

CCMB80 buffer

• 10 mM KOAc pH 7.0
• 80 mM CaCl₂.2H₂O
• 20 mM MnCl₂
• 10 mM MgCl₂.6H₂O
• 10 % glycerol

Procedures

1. Set an ON starter culture by inoculating 5 ml LB and incubating ON in 37 °C with 250 rpm rotary shaking.
2. Inoculate 250 ml new LB with 2 ml from the ON culture and grow in 30 °C, 250 rpm until an OD₆₀₀ of ≈0.3.
   • Use a large, 1 l E-flask.
3. Spin down cells at 3000 x g for 10 min in 4 °C.
4. Remove supernatant and resuspend cells in 80 ml ice-cold CCMB80 buffer. Keep cells on ice for 20 min.
5. Spin down cells again with same settings. Resuspend in 10 ml new CCMB80 buffer and keep on ice for 20 min.
6. Aliquot 100 μl samples of competent cells into 1.5 ml vials and store in -80°C, or transform immediately.

Transformation (Nina & Johan)

From NEB 5-alpha competent E.coli (High Efficiency) NEW ENGLAND BioLabs

1. Thaw a tube of NEB 5-alpha competent E.coli cells on ice for 10 min.
2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix the cells and DNA.
3. Place the mixture on ice for 30 min.
4. Heat shock at 42°C for 30 sec.
5. Place on ice for 5 min.
6. Pipette 950 μl of room temp. SOC into the mixture.
7. Place at 37°C for 30-60 min, 250 rpm.
8. Warm selection plates to 37°C.
9. Perform 10-fold serial dilutions in SOC (if necessary)
10. Spread 50-100 μl of each sample onto a selection plate and incubate overnight at 37°C.

Transformation (Andreas & Mimmi)

1. Add 1 μl plasmid to 100 μl thawed, competent cells of choice. Hold cells on ice for 30 min.
2. Heat-shock cells for 55 sec in 42 °C. Return to ice.
3. Add 900 μl LB medium and grow cells in 37°C, 250 rpm for 1 hour.
   • This allows cells to start expressing antibiotic resistance gene(s).
4. Spin down cells at full speed (≈13.000 x g) for 15 sec.
5. Remove 900 μl from the supernatant and gently resuspend pellet in remaining 100 μl.
6. Plate 100 μl cells onto an LB agar plate with appropriate antibiotic(s).
7. Incubate in 37 °C ON.

Quick transformation (Andreas & Mimmi)

1. Add 1-3 μl plasmid to 100 μl thawed, competent Top10 cells. Hold cells on ice for 5 min.
2. Heat-shock cells for 30 sec in 42 °C. Return to ice.
3. Plate cells onto a pre-heated (37 °C) LB agar plate with appropriate antibiotic(s).
4. Incubate in 37 °C ON.

Colony PCR verification (Andreas & Mimmi)

1. Pick four colonies and resuspend each in 10 μl LB.
   • Let incubate in RT while preparing PCR tubes.
2. Prepare each illustra PuReTaq Ready-To-Go PCR (GE Healthcare) tube as follows:
   • 1.0 μl 10 μM forward primer
   • 1.0 μl 10 μM reverse primer
   • 22.5 μl dH₂O
   • 0.5 μl cell suspension (template DNA) to a final volume of 25 μl. Vortex to mix.
3. Run PCR amplification.
   • Denaturation: 95 °C - 10 min
   • 30 cycles
   1. Denaturation: 95 °C - 30 s
   2. Annealing: 55 °C - 30 s
   3. Elongation: 72 °C - Calculate from expected sequence length, ≈1 min/kb
   • Elongation: 72 °C - 10 min
4. Analyze PCR products by agarose gel electrophoresis.

Site-directed mutagenesis

Based on the QuikChange® Site-Directed Mutagenesis Kit

• Design two complimentary oligonucleotides containing the desired mutation with https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&SubPageType=ToolQCPD&PageID=15

1. Prepare PCR (amounts for 100 µl reaction)
   • 10 ng dsDNA template
   • 125 ng primer #1
   • 125 ng primer #2
   • 84 µl ddH2O
   • 2 µl 10 mM dNTPs
   • 10 µl 10x Pfu reaction buffer
   • 2 µl Pfu Turbo DNA polymerase
2. Run PCR
   1. 95 °C - 30 sec
   2. 22 cycles of
      1. 95 °C - 30 sec
      2. 55 °C - 1 min
      3. 68 °C - 1 min/kb plasmid
   3. 4 °C - ∞
3. Make sure the reaction is ≤ 37 °C before proceeding
4. Add 2 µl (for 100 µl reaction) of Dpn I restriction enzyme and mix thoroughly
5. Spin down for 1 min in microcentrifuge
6. Immediately incubate at 37 °C for 1 hour or more (more gives less background)

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SDS-PAGE mixtures (Nina & Johan)

From Robert Daniels at the Department of Biochemistry & Biophysics Stockholm University

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Mini prep(Nina & Johan)

Based on the QIAprep Spin Miniprep Kit Using a Microcentrifuge

1. Centrifuge sample in 4 °C, 10 min, 4000 rpm and resuspend pelleted bacterial cells with Buffer P1 (250 ul/5ml bacterial sample) and transfer to a microcentrifuge tube.

2. Add 250 ul buffer P2, invert the tubes 4-6 times.

3. Add 350 ul buffer N3, invert the tubes 4-6 times with powerful strokes.

4. Centrifuge 10 min, 13000 rpm in a table-top microcentrifuge.

5. Transfer the supernatant to the QIAprep spin column.

6. Centrifuge for 1 min, 13000 rpm. Discard the flow-through.

7. Add 0.5 ml buffer PB and centrifuge for 1 min, 13000 rpm. Discard the flow-through.

8. Add 0.75 ml buffer PE and centrifuge for 1 min, 13000 rpm.

9. Discard the flow-through and centrifuge again the same way.

10. Place the column in a clean 1.5 ml microcentrifuge tube. Add 35 ul water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min, 13000 rpm.

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Agarose gel clean up (Nina & Johan)

Based on the QIAprep II Handbook

IgG protease activity assay

Based on an experiment by [http://www.ncbi.nlm.nih.gov/pubmed/20441890 Abuknesha, et al (2010)].

Materials

• 10 ml culture tubes
• 200 ml E-flasks
• 10 ml Falcon tubes
• IPTG
• Protease dilution buffer
• Eppendorf tubes
• [http://en.wikipedia.org/wiki/McFarland_standards 0.5 McFarland standard]
  • 0.05 ml 1 % BaCl₂
  • 9.95 ml 1 %/0.18 M H₂SO₄
• [http://openwetware.org/wiki/PBS Phosphate buffered saline (PBS), pH 7.4]
• Mouse IgG-Agarose (1 mg IgG/ml) (Sigma-Aldrich)
• Anti-mouse IgG peroxidase conjugate (Sigma Aldrich)
• SureBlue™ TMB Microwell Peroxidase Substrate

Procedures

Day 0

1. Set ON cultures in 5 ml LB + 100 μg/ml Amp, 37 °C, 225 rpm
   • BL21 IgGp
   • BL21 SOD (or other negative control)

Day 1

1. Inoculate 200 μl of each ON culture (x2 for IgGp) into 20 ml fresh LB with 100 μg/ml Amp. Grow at 37 °C, 225 rpm until an OD₆₀₀ of ≈0.5.
2. Induce protein expression by adding IPTG to a final concentration of 0.3 mM (e.g. 60 μl 0.1 M IPTG) to one of each culture type. Leave the second IgGp culture uninduced (negative control). Continue incubation for two hours.
3. Transfer cultures to 10 ml Falcon tubes and spin down cells at 4,400 x g for 10 min.
4. Resuspend enough pellet in 5 ml sterile PBS to yield a cell density of 1.5 x 10⁸ CFU ml^-1 according to the McFarland standard 0.5. Make a 1000x dilution of each culture with PBS to 1.5 x 10⁵.
5. Sonicate cells and spin down cell debris at 4,400 x g for 10 min. Recover and save supernatants for protease assay.
   • Uninduced IgGp
     • 1.5 x 108 CFU ml-1
     • 1.5 x 105 CFU ml-1
   • Induced IgGp
     • 1.5 x 108 CFU ml-1
     • 1.5 x 105 CFU ml-1
   • Induced SOD
     • 1.5 x 108 CFU ml-1
     • 1.5 x 105 CFU ml-1
6. Prepare IgG peroxidase dilutions by diluting the stock solution in PBS to 1:500 and 1:1000.
7. Apply 30 μl IgG-Agarose suspension to 14 Eppendorf tubes, two for each protein extract dilution and two negative controls.
8. Bind secondary antibodies by adding 100 μl anti-mouse IgG peroxidase, each dilution (1:500, 1:1000) to 7 tubes. Allow antibody binding by incubating at room temperature with tilt mixing for 30 min.
9. Spin down IgG-Agarose at 13,000 x g for 3 min and wash 3 times with 200 μl PBS. Spin down again and remove supernatant.
10. Add 100 μl of the protease extracts to the corresponding tubes (12 tubes). Add 100 μl PBS to the remaining two negative control tubes. Incubate at 37 °C ON (≈16 h) with tilt mixing.

Day 2

1. Spin down the IgG-Agarose at 13,000 x g, 3 min. Transfer 80 μl of each supernatant (no pellet!) to new Eppendorf tubes.
2. Add 100 μl Sure Blue™ peroxidase substrate solution. Incubate in room temperature and tap gently until a blue color develops (≈30 min).
3. Stop reaction by adding 100 μl 1 M HCl.
4. Read color intensity/absorbance at 450 nm.