Team:Marburg/Notebook:October

From 2013.igem.org

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<p>Result: 5.16 µg/µl protein were extracted.</p>
<p>Result: 5.16 µg/µl protein were extracted.</p>
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    <legend>SDS-PAGE and Western blot</legend>
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    <div class="investigator">
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<span class="inv">Investigator:</span>
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<span class="inv-names">Domenica, Franzi and Dominik</span>
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    <div class="aim">
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<span class="aim">Aim:</span>
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<span class="aim-desc">Quantify the amount of eGFP produced under different light conditions.</span>
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<p>5 µg (ball mill) and 10 µg (sonication) protein was used for a SDS-Page, after that, a Western Blot was performed (overnight, 4°C, 30V).</p>
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Revision as of 18:12, 28 October 2013

Notebook: October Next Previous

19.10.2013

Light-inducible promoter
Investigators: Domenica and Dominik
Aim: Characterization of the light-inducible promotor PfcpB (BBa_K1071003).

Erlenmeyer flask with 50 mL P. tricornutum culture was coated with aluminium foil to prevent induction of PfcpB.

21.10.2013

Cell disruption by means of glass beads
Investigator: Dominik
Aim: Test of glass beads disruption method.

  • 4 ml cell culture was harvested.
  • Pellet was dissolved in 1 mL PBS.
  • 0.5 ml glass beads were added.
  • Tubes were shaken for 3 min → color shift from brown to green.
  • Trichloracetic acid (TCA) precipitation was performed.

Composition of the phosphate-buffered saline (PBS) buffer:
Sodium chloride   137 mM
Potassium chloride   2.7 mM
Na2HPO4   10 mM
KH2PO4   1.8 mM

Cell disruption by means of ball mill
Investigator: Domenica
Aim: Test of ball mill disruption method.

  • 4 ml cell culture was harvested.
  • Dilution in 300 µL 8M urea buffer.
  • Shock freezing of suspension by means of liquid nitrogen.
  • Two steel balls were added → Tube was shock freezed again.
  • Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.

Composition of the urea buffer (for 40 ml):
Urea 8 M   19,22 g
2 M Tris-HCl pH 6,8   4 ml
0,5 M EDTA   8 µl
10 % SDS   20 ml
Bromophenol blue   12 mg

Cell disruption by means of sonication
Investigator: Franzi
Aim: Test of sonication disruption method.

  • 4 ml cell culture was harvested (5 min centrifugation at full speed).
  • The supernatent was discarded and the pellet frozen in liquid nitrogen.
  • The frozen pellet was resuspended in 0.68 ml IP buffer and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high).
  • Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.
  • 1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice.
  • The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone.
  • The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).

Composition of the immuno precipitation (IP) buffer:
HEPES-KOH buffer pH 7.5   50 mM
Sodium chloride   150 mM
EDTA   1 mM
Triton X 100   1 %
Sodium deoxycholate   0.1 %
Sodium dodecyl sulfate (SDS)   0.1 %

SDS-PAGE
Investigator: Dominik
Aim: Identification of the most efficient disruption technique.

20 µl of each cell suspension obtained from the three different disruption techniques were load on a 12 % SDS gel.

Separation gel
1 M Tris-HCl pH8,8   2,25 ml
Aa/Bis 30:0,88   2,4 ml
H2O   1,2 ml
10 % SDS (w/v)   75 µl
10 % APS   100 µl
TEMED   10 µl

Stacking gel
1 M Tris-HCl pH6,8   375 µl
Aa/Bis 30:0,88   500 µl
H2O   2050 µl
10 % SDS (w/v)   30 µl
10 % APS   50 µl
TEMED   7,5 µl

SDS gel
gel-electrophoresis-image

Gel substances

  • 12 % Acryl amide gel
  • 6 µl Prestained Page Ruler (Thermo Scientific)

    • Lane   Content   Expectations
    2   Ball mill + protease inh.   26 kDa (lc) + 55 kDa (hc)
    3   Ball mill   26 kDa (lc) + 55 kDa (hc)
    4   Glass beads + prot. inh.   26 kDa (lc) + 55 kDa (hc)
    5   Glass beads   26 kDa (lc) + 55 kDa (hc)
    6   Sonication (total)   26 kDa (lc) + 55 kDa (hc)
    7   Sonication (SN)   26 kDa (lc) + 55 kDa (hc)
    8   Sonication (P)   26 kDa (lc) + 55 kDa (hc)

Result: The highest amounts of proteins were gained using sonication and the ball mill.

Test of antibody production
Investigator: Dominik
Aim: Preparation for Western Blotting.

To demonstrate the production of antibodies in the induced cultures 2.1 and 4.2 a Western Blot should be performed. For this purpose, the cells were harvested and the pellet was separated from the supernatant. After that, a TCA precipitation was performed. All samples were stored at -20°C.

22.10.2013

Cell disruption by means of sonication
Investigator: Franzi
Aim: Find the best buffer for sonication disruption method.

To examine whether the PBS buffer or the IP buffer is best for disruption by means of sonication, we performed a sonication with both buffers. Afterwards a TCA precipitation was made and a SDS-PAGE was carried out.

Result: We gained higher amounts of protein by using the IP buffer. Consequently, all following cell disruptions by sonication were performed with IP buffer.

Light-inducible promoter
Investigators: Domenica and Dominik
Aim: Characterization of the light-inducible promotor PfcpB (BBa_K1071003).

After two days of incubation in darkness, the culture was divided in 5 samples, which were incubated under 5 different light conditions: normal light, green light, red light, blue light (50 µE each) and darkness. The division was realised in darkness to prevent induction of the light-inducible promoter. OD (600 nm): 0.450.

   lightning chamber
Test of antibody production
Investigator: Dominik
Aim: Western Blot of samples before and after induction.

The samples that were taken the previous day and the days before induction with nitrate were used for a 12 % SDS gel (see above) and subsequent Western blotting on a nitro cellulose membrane. To accomplish this four Whatman filter papers were soaked into membrane buffer and a nitro cellulose membrane, also soaked in membrane buffer, were stacked. On this the SDS gel was placed and covered with with four Whatman filter papers soaked with gel buffer. Then the proteins were transferred with 0.8 mA/cm2 for two hours.

To prevent unspecific binding of the first antibody, the membrane was rotated for at least one hour in 5 % milk powder in TBST at 4 °C. The primary antibody was dissolved in 2 % milk powder in TBST (dillution: 1:500 anti GFP, anti IgG 1:10,000, all antibodies obtained by Santa Cruz biotechnology) and added to the membrane. Afterwards it was washed two times in TBST for five minutes. The secondary antibody was dissolved in 2 % milk powder in TBST (anti anti GFP 1:10,000, anti anti IgG 1:10,000), added to the membrane and incubated for one hour. Afterwards it was washed with TBST for three times for five minutes each. After incubation with a detection agent, containing Luminol, p-Coumaric acid and H2O2, the detection was performed with a Chemocam Professional from INTAS science imaging.

Composition of the Tris-buffered saline-tween (TBST) buffer:
Tris-HCl pH 7.6   50 mM
Sodium chloride   150 mM
Tween 20   0.05 %

    Sample preparation:
  • Resuspend pellet in 1 ml water
  • Measure OD (600 nm)
  • Take the volume corresponding to a certain OD
  • Protein preparation of the taken sample with the predefined OD
    • Cell culture is centrifuged for 5 minutes (full speed)
    • Discard supernatant
    • Add 150 µl sodium hydroxide
    • Incubate 10 minutes on ice
    • Add 850 µl bidest. water
    • Add 200 µl 70 % TCA and incubate for 20 min on ice
    • Centrifuge for 15 minutes at 4 °C (20000xg)
    • Wash pellet with aceton and centrifuge for 15 minutes at 4 °C
    • Repeat the latter step until the chlorophyll is washed out
    • Dry pellet at room temperature
    • Resuspend in 8 M urea buffer
    • Shake the samples for 15 min at 60 °C
    • Load samples on a 12 % SDS gel
  • Load samples on the gel
  • Run gel for at least 1 h (120V)
  • Mini-Protean Tetra Cell (Biorad)
  • Equilibrate the gel in Towbin transfer buffer (25 mM Tris, 192 mM glycine pH 8.3, 20 % MeOH) for 10 min
  • Blotting on a nitrocellulose membrane using the STANDARD SD transfer protocol from Biorad preprogrammed protocols (Trans-Blot Turbo Blotting System)
  • Submerge blot in 4 % milk powder in TBST
  • Incubate for 2 h at 4 °C
  • Incubate membrane for 1-2 h with the milk and the primary antibody against GFP and tubulin (dilution: 1:1000 GFP, 1:2000 tubulin)
  • Wash membrane in TBST for 10 min (3 times)
  • Incubate for 1 h at 4 °C with the secondary antibody in milk-TBST (goat anti mouse HRP)
  • Wash for 10 min at 4 °C in TBST (3 times)
  • Detection in western imager station (chemocam)
    • ECL solution: incubate membrane for 1 min in ECL solution
Western blot 1
gel-electrophoresis-image In these samples that were taken before the induction, bands at 80 kDa could be detected. They could be due to unspecific bounding of the antibodies.
The second blot, containing the samples that were taken after induction, failed because of a lack of enough transfer buffer.
Additionally, due to insufficient blocking steps, much background signal was observed. For that reason, no statements about the production of antibodies can be made.

23.10.2013

Test of antibody production
Investigators: Dominik, Franzi and Domenica
Aim: Harvesting, cell disruption and TCA prepipitation.

10 mL of the culture were harvested for cell disruption with sonication (see protocol below), 1 mL for cell disruption with the ball mill, and 1 mL as a reserve. Cells were disrupted with sonication and the ball mill. Optical density (at 600 nm) was measured for each sample.

Condition   OD600  
Light   0.525  
Darkness   0.410  
Blue light   0.576  
Red light   0.505  
Green light   0.552  

Sonication
Investigators: Franzi, Marco
Aim: Quantification of the light-inducible promoter PfcpB (BBa_K1071003)
→ Cell disruption, protein precipitation and SDS-PAGE.

10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.

Frozen cell-pellets were resuspended in 1,7 ml IP buffer and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.

1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).

10 µg of each sample were used for SDS-PAGE (Westernblot).

Amido black assay
Investigators: Franzi, Dominik, Domenica
Aim: Quantification of PfcpB → Quantification of protein amount.

5 µl of protein (in urea buffer) from sonication and ball mill disruption were used.

    The following protocol was used:
  • 195 µl H2O
  • 5 µl sample (or 5 µl H2O as negative control)
  • 800 µl amido black solution (0.1 % amido black solution, 90 % MeOH, 10 % acetic acid (100 %))
  • Invert 3 times → precipitation of protein-amido black complex
  • Centrife: 10 min @ 14,000 rpm
  • Discard supernatant
  • Add 1 ml washing solution (90 % EtOH, 10 % acetic acid (100 %))
  • Centrife: 10 min @ 14,000 rpm
  • Discard supernatant
  • Add 1 ml 0.2 M NaOH → complete bleaching of the pellet

The protein concentration can be determined photometrically by measuring the absorbance at 615 nm and by comparing the values with a standard curve (created with different amounts of BSA).

    Measured protein concentrations for sonication samples:
  • Dark: 2.10 µg/µl
  • Red: 2.49 µg/µl
  • Green: 2.80 µg/µl
  • Blue: 3.23 µg/µl

    Measured protein concentrations for ball mill samples:
  • Light: 0.517 µg/µl
  • Dark: 0.314 µg/µl
  • Red: 0.639 µg/µl
  • Green: 0.385 µg/µl
  • Blue: 0.619 µg/µl
Protein extraction from herbal leafs
Investigators: Domenica, Franzi and Dominik
Aim: Protein extraktion (positive control).

As a positive control, we extracted a protein-coding gene, cry-gfp, out of Arabidopsis landsberg erecta, which produces GFP as well as tubulin. For this purpose, 228 mg leafs were mixed with 114 µL PEG extraction buffer. Following this, the cells were disrupted with a Polytron PT 1200E Blade-type homogenizer (Brinkmann) and 5 min in a sonication bath. After centrifugation (15 min, 20000g, 4°C), the supernatant was used as protein extract. The concentration of protein was determined using a Bradford assay (Roti Quant, Roth).

Composition of the polyethylene glycol (PEG) extraction buffer:
Tris-HCl pH 7.8   0.5 M
Triton X 100   2 %
Magnesium chloride   20 mM
Phenylmethylsulfonyl fluoride (PMSF)   1 mM
Ethylenediaminetetraacetic acid (EDTA)   1 mM
Polyvinylpolypyrrolidone (PVPP)   1 %

Result: 5.16 µg/µl protein were extracted.

SDS-PAGE and Western blot
Investigator: Domenica, Franzi and Dominik
Aim: Quantify the amount of eGFP produced under different light conditions.

5 µg (ball mill) and 10 µg (sonication) protein was used for a SDS-Page, after that, a Western Blot was performed (overnight, 4°C, 30V).

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