Team:Freiburg/Labjournals/ProtPur/July
Protein Purification
30.07.2015
phyA purification
- o/n culture 5ml was inoculated and set at 37°C (Amp+Gent)
purification of pET_1003; pET_1103; pET_1703 and pIG15_1301; pIG15_1501
- additional to the purification protocol the flow-through of each sample was incubated o/n with Ni-NTA beads to test wether there is still target protein in the sample.
- the next day beads were washed with 3 mL washing buffer and eluted in 2 fractions
- fractions are labeled E6 and E7 on the gels
Results
- Determination of protein concentrations by Amido-Black
- from the slope and the intercept of the standard curve an amount of protein was computed for every sample measured. That amount was compared with the results from the nanodrop measurement.
Protein concentration from the amidoblack and the nanodrop measurements were compared. Values that are not covered by the amidoblack standard curve are marked in red
| Abs | Protein amonut [ng] | conc [mg/mL] | conc nanodrop [mg/mL] | difference | ||
|---|---|---|---|---|---|---|
| 1003 | E1 | 0,517 | 15,77556326 | 0,788778163 | 0,985 | 0,196221837 |
| E2 | 0,389 | 11,67157712 | 0,583578856 | 0,743 | 0,159421144 | |
| E3 | 0,05 | 0,802426343 | 0,040121317 | 0,078 | 0,037878683 | |
| 1103 | E1 | 0,731 | 22,63691508 | 1,131845754 | 2,937 | 1,805154246 |
| E2 | 1,033 | 32,31975737 | 1,615987868 | 3,755 | 2,139012132 | |
| E3 | 0,101 | 2,437608319 | 0,121880416 | 0,509 | 0,387119584 | |
| 1301 | E1 | 0,675 | 20,84142114 | 1,042071057 | 3,648 | 2,605928943 |
| E2 | 0,352 | 10,48526863 | 0,524263432 | 1,613 | 1,088736568 | |
| E3 | 0,08 | 1,764298094 | 0,088214905 | 0,28 | 0,191785095 | |
| 1501 | E1 | 3,006 | 95,57885615 | 4,778942808 | 5,462 | 0,683057192 |
| E2 | 2,393 | 75,92461005 | 3,796230503 | 6,713 | 2,916769497 | |
| E3 | 0,462 | 14,01213172 | 0,700606586 | 1,083 | 0,382393414 | |
| 1703 | E1 | 1,014 | 31,71057192 | 1,585528596 | 2,212 | 0,626471404 |
| E2 | 0,348 | 10,35701906 | 0,517850953 | 1,009 | 0,491149047 | |
| E3 | 0,058 | 1,058925477 | 0,052946274 | 0,2 | 0,147053726 |
- SDS-PAGE-Analysis
Salmonella Antigen 63kDa + Antibody 35kDa
Hepatitis C Core Protein (pET_1003)22kDa
Clostridium tetani (pET_1103)50kDa
HIV (pET_1703) 20,5kDa
29.07.2015
Purification of phyA
- phyA1-406, #1652
- N-terminal phA fragment in pGEX was transformed in the Arctic strain and plated out on Amp+Gentamycin, o/n 37°C
purification of pET_1003; pET_1103; pET_1703 and pIG15_1301; pIG15_1501
- the pellets of 1 liter cultures were resuspended and sonificated according Cell disruption
- the purification was performed according to the Protein Purification on gravity flow columns
- with this purification we wanted to increase the protein concentration by the double amount of culture
- for analysis, samples of all steps during the purification for the SDS-PAGE-Analysis; WesternBlot; and for the determination of the concntration by the Amido-Black protocol.
- the elution were handed over to the iRIf and Surchem group
Results
To verify the antigen-antibody interaction we made a dot blot of each antigen with the specific antibody (LISTE ERSTELLEN) and the His-conjugated antibody from Qiagen. Additional we performed western blots according to Western Blot towbin et al.. This western blot should confirm the interaction of the new polyclonal antibodies for HCV and HIV. For C.tetani we only have a monoclonal one. Also the gels were stained with coomassie after the blotting step onto the PVDF membrane. The blocking step was performed with 5% milk in TBS-T. The primary polyclonal antibodies were 1:500 diluted in TBS-T containing 5% BSA. The primary antibody was incubated o/n, 4°C.
Dot blot
The dot blot for the specific antibodies looks very promising. But the western blots show a very unspecific binding of the polyclonal antibodies. Unfortunately the highest signal of the antibody-binding is not the target protein.
Salmonella (pIG15_1501 and pIG15_1301)
Clostridium tetani (pET_1103)
Hepatitis C Core Protein (pET_1003)
HIV poI/gag/env(pET_1703)
it is obvious, that also the his-conjugated antibody show unspecific binding. However in further experiments the presence of the target proteins was verified. Useful for comparison are the western blots of pET_1003 and pET_1703 on 16.07.2015; pET_1103 on 14.07.15; pET_1003 and pET_1703 on 10.07.2015 and pIG15_1501 and pIG15_1301 on 02.07.15. We are always working with the same colony out of the glycerol stock with double selection: Ampicilin for the plasmid and chloramphenicol for the E.coli strain
28.07.2015
Purification of phyA
- got 5 µL of DNA (phyA-GST-His in pGEX #30918) from Philipp
- transformed Top10 cells for test digest and BL21 for purification according to transformation protocol
- growth o/N @ 37°C on plates
- put DNA into fragments-box in -20°C freezer
- the plasmid with the N-terminal phyA fragement was only transformed. for further experiments we are using another N-terminal fragement in pGEX-plasmid. For this protein the E.coli strain „Arctic“ is recommended
27.07.2015
pET_803, pET_1003, pET_1103, pIG_1301, pIG_1501 & pET_1703
- 1 liter of LB-medium was inoculated with the particular o/n culture, except for pIG15_1501. Here we only used 500ml LB-medium.
In this case we do not have to increase the culture due to the fact of high protein concentrations.
- the cultures were set to 37°C, 180rpm
- after an OD600 ~ 0.5 the cultures were induced with IPTG; the best IPTG concentration was tested in several overespression test and is recorded for each strain in the masterplan.
- cells were harvested by centrifugation (5000 g, 30 min, 4°C). The supernatant (LB-medium) was discarded and the pellet was washed in 10ml lysis buffer. To store the pellet, the 10ml (lysis buffer + resuspended cells) were centrifuged again (10 000rpm, 30min, 4°C) and frozen in the -20°C freezer.
26.07.2015
pET_803, pET_1003, pET_1103, pIG_1301, pIG_1501 & pET_1703
- 5ml o/n cultures of each plasmid transformed in a specific E.coli strain
- exisitng glycerol stocks were striked out on Amp+Cml plates for further o/n cultures
25.07.2015
Purification of pET_803, pET_1003, pET_1103, pIG_1301, pIG_1501 & pET_1703
Experiment
- the blots were washed 3 times 10 min in TBS-T and incubated for 1h with secondary AB (coupled to HRP)
- 2x 10min wash with TBS-T
- Chemiluminscence detection with ECL-reagent
Results
25.07.2015
Purification of pET_803, pET_1003, pET_1103, pIG_1301, pIG_1501 & pET_1703
- The PVDF-membranes were washed 3 times 10min in TBS-T and incubated o/n in primary antibody solution
- The solutions for anti-His-Tag, anti-HIV and anti-HCV were prepared freshly whereas for C. tetani an already prepared solution was used
| construct | antibody used |
|---|---|
| pET_1103 | mouse anti-tetanus 1:1000 |
| pET_1003 | rabbit anti-HCV 1:2500 |
| pET_1703 | mouse anti-HIV 1:1000 |
| all constructs | mouse anti-Histag 1:1000 |
24.07.2015
Purification of pET_803, pET_1003, pET_1103, pIG_1301, pIG_1501 & pET_1703
- 12.5 % SDS gels were casted and protein samples from the day before were loaded (15 µL each)
- Gel was run @ 0.03 A per gel for 1:30 h
- Staining with coomassie solution and direct destaining according to microwave protocol
23.07.2015
Purification of pET_803, pET_1003, pET_1103, pIG_1301, pIG_1501 & pET_1703
Experiment
- o/n grown samples for pET_1003, pIG_1301 and pET_1703 were harvested by centrifugation (4300 g, 30min 4°C)
- pellets of all condiitions (incl. those harvested the day before) were resuspended in 10 mL Lysis buffer per culture condition (using 3 glass beads per falcon to aid resuspension)
- Suspensions were sonified (5min, 60% power, cycle 6)
- aliquot was taken for later SDS-PAGE and dotblot analysis
- cell debris was removed by first centrifugation (10000g, 4°C, 30 min)
- supernatant was further cleared from particels by second centrifugation (16000g, 4°C, 20 min)
- supernatant was taken
- supernatant was loaded on an equilibrated Ni-NTA column each and purified with 3 mL washing steps and 5x 500 µL elution volume
- aliquots were taken from the eluate fractions
- eluate fractions were pooled and loaded on a desalting column equilibrated with lysis buffer before
- for pET_803 - pET_1103 the whole 6 mL elution from the desalting column was pooled for the other half the elution was split into 3 fractions
- a dot blot was prepared with the respective samples lysates, elutions 2 and desalted fractions
- aliquots for SDS-PAGE were mixed with 2.5x SDS loading dye and boiled @ 95°C for 10 min
- protein concentration in the final samples was determined via nandrop analysis befor ethe samples were handed over to the SurChem group for spotting on thier Ni-NTA functionalized glass slides.
Results
For half the samples all 6 mL elution volume were pooled whereas for the other half the elution volume was split into 3. In bold the samples taken for iRIf spotting are marked.
| E1 | E2 | E3 | |
|---|---|---|---|
| 803 | 0.13 mg/mL | ||
| 1003 | 0.13 mg/mL | ||
| 1103 | 0.55 mg/mL | ||
| 1301 | 0.15 mg/mL | 0.4 mg/mL | 0.0 mg/mL |
| 1501 | 1.06 mg/mL | 3.24 mg/mL | 0.4 mg/mL |
| 1701 | 0.25 mg/mL | 0.36 mg/mL | 0.1 mg/mL |
22.07.2015
- 500ml LB-medium (Cml+Amp) was inoculated with 5 ml o/n culture of pET10;08;17 and pIG15_13 + 15, respectively.
- after an OD600 of 0.5 each culture was induced with the distinct IPTG concentration according to the masterplan.
20.07.2015
pET_1003, pET_1103 & pET_1703
- Pellets of each strain were resuspended in 2x 5mL Lysis buffer by using 3 glass beads and 5 mL buffer per Falcon tube, shaking the tube until pellet was resuspended and transferring the suspension with the beads to the next tube
- Cells were broken up by sonification (5min, cycle 6, 80% power)
- 1:1000 300 mM PMSF was added
- Cell debris was removed by centrifugation (30min, 10000g, 4°C)
- supernatant was transferred into new falcon tubes
- smaller cell debris was removed by a 2nd centrifugation (160000g, 20min, 4°C)
- cell lysates were loaded onto Ni-NTA agarose columns and purified as described
- elution was carried out in 5 fractions with 500 µL elution buffer each
- aliquots for SDS-PAGE and Western Blot were taken and protein concentrations were determined
- purified proteins were handed over to the iRIf group
For further analysis we took samples of each fraction and made a wester blot using the specific antibodies of each antigen. We also analysed by a 12,5% gel our efficiency of the protein purification.
21.07.2015
Western Blot: The western Blot was performed according to the protocol WB Towbin et al. The specific antibodies were used in the dilutions:
- anti Clostridium tetani (mouse) 1:1000, clone 15
- anti Hepatitis C Core Protein (rabbit) 1:2500
- anti HIV (mouse) 1:1000, gp41 DDX1306
- the secondary AB was used in a 1:2500 dilution, recommendation of the company
- each antibody was incubated for 1h at RT
Results
19.07.2015
pIG15_1301 C43 and pIG15_1501 Rosetta protein purification
- frozen pellets were resuspended in 10ml/g pellet lysis buffer: We changed the lysis buffer. Now, we are using 20mM Tris, 150mM NaCl, 10mM Imidazole, pH 7,5 without any Phosphate. We had problems with denaturated protein after thawing. The reason could be the phosphate containing buffer, which results in a fluctuating pH-range during the thawing.
- resuspended cells were sonifigated 5min, 6cycles, 80%
- afterwards disrupted cells were centrifuged for 30 min, 10 000xg, 4°C
- the supernatant was again centrifuged to get rid of contaminations. 20min, 11000xg, 4°C
- the disposable columns were washed with degased water and the 50% slurry (1ml) was pipetted on the column.
- the equilibration of the Ni-NTA was performed with 5 column volumes
- afterwards the supernatant was loaded on the column, the flow-through was collected
- when the binding process was completed a washing step with 20 column volumes was performed [washing buffer: 20mM Tris, 150mM Nacl, 20 mM Imidazole, pH 7,5]
- the elution of the target proteins was performed with the elution buffer [20mM Tris, 150mM Nacl, 500 mM Imidazole, pH 7,5]
- stepwise elution was performed with 500ul fractions five times.
- the different fractions as well as the washing step and flow-through was loaded on a 12,5% SDS-PAGE gel for analysis
- the columns were treated with a total elution buffer [20mM Tris, 150mM Nacl, 800 mM Imidazole, pH 7,5]
- afterwards washed with degased pure water (10x column volumnes)
- and stored in 20% EtOH, 4°C
Results
- the gels show a good enrichment of both proteins and were handed out to the iRIf group on the same day.
- we recommended elution 3 of pIG15_1501 and elution 2 of pIG15_1301
- western blot for these proteins was performed on 02.07.2015
18.07.2015
pIG15_1301 C43 (RJ)
- harvested cells in C43 from o/n, 30°C and froze them on -20°C
pIG15_1501 Rosetta (RJ and LS)
- glycerol stock of 1 ml of o/n culture
- inoculated 500 ml LB Amp/Cml with 5ml of o/n culture
- cells grew too much (OD ~ 1.2) → did a 1:2 dilution and measured again (OD ~ 0.6)
- waited 5 minutes and induced with 1 mM IPTG
- after 3 h induction the cells were harvested
- Mini from Top10 plate to have more plasmid for future trafos, concentraction: 31 ng/µl
16.07.2015
pET_1003 & 1703 (JB)
- 5 mL o/n culture were suspended in 500 mL LB medium (Amp, Cml)
- Cultures were grown on 37°C until OD_600 = 0.5
- Induction with 0.1 mM or 1 mM for 1003 and 1703 respectively
- Growth o/n
general labwork (JB)
- set up 10x 1 mL new ampicillin aliquots according to Antibiotika Stocks protocol
- froze aliquots in -20°C
- prepared new Tris-buffers for purification according to Tris purification buffers protocol
- 1 L Lysis buffer
- 0.5 L Wash buffer
- 0.1 L Elution buffer
- 50 mL Total elution buffer
pIG15_1301 (RJ)
- inoculated 5 ml LB Amp/Cml with Kryostock of pIG15_1301 C43
- growth o/n
15.07.2015
pET_1003; pET_1703
In this experiment it should be varified whether the target proteins are soluble or not.
- the different 15 ml samples were sonificated (5min, 6cycles, 70%)and PMSF was added
- afterwards the samples were centrifugated 10 000xg, 30 min, 4°C
- the supernatant was seperated from the pellet. The corresponding pellet was resuspendend in 5ml lysis buffer (same volume as the supernatant)
- for further analysis of each fraction 12ul was taken for western blotting against the his-tag.
pET_1003; pET_1703 Western Blot
- 12,4% SDS-PAGE gel for western blotting
- semi-dry blotting was performed with PVDF membrane for 1h, 0.8mA/cm2 gel
- the membrane was washed twice for 10min in TBS buffer at RT
- Incubation for 1h in blocking solution at RT (special blocking solution of Qiagen; 1xblocking buffer, 0.5% blocking powder, 0.1% Tween)
- afterwards membrane was washed for 10 min in TBS-Tween at RT
- Following the incubation in Anti-His HRP Conjugate (1:1000 in blocking buffer)
- membrane was washed twice for 10 min in TBS-T
- Chemiluminescent detection was performed with peroxide solution and luminol enhancer solution
Result
14.07.2015
pET_1103 Western Blot with anti- //Clostridium tetani// toxoid
This experiment should verify the interaction between the antigen Clostridium tetani TeNT_Hc [pET_1103] (50 kDa) and the anti-clostridium tetani toxoid monoclonal antibody (mouse) we got sponsored from the AntibodyShop.
- 12,5% SDS-PAGE gel analysis
- semi-dry blotting was performed with activated PVDF membrane 1h, 0.8mA/cm2 gel [membrane was cutted in two pieces for different antibodies]
- blocking in 5% milk powder in TBS-T for 1h at RT
- membrane was washed twice with TBS-T
- 1st antibody anti-clostridium tetani toxoid was incubated o/n, 4°C (1:1000 in TBS-T)
- membrane was washed twice with TBS-T
- 2nd antibody was incubated for 1h at RT, anti-mouse HRP (1:5000 in TBS-T)
- membrane was washed twice with TBS-T
- Chemiluminescent detection was performed with peroxide solution and luminol enhancer solution
For the anti-His Tag antibody the protocol according to Qiagen was performed:
- the membrane was washed twice for 10min in TBS buffer at RT
- Incubation for 1h in blocking solution at RT (special blocking solution of Qiagen; 1xblocking buffer, 0.5% blocking powder, 0.1% Tween)
- afterwards membrane was washed for 10 min in TBS-Tween at RT
- Following the incubation in Anti-His HRP Conjugate (1:1000 in blocking buffer)
- membrane was washed twice for 10 min in TBS-T
- Chemiluminescent detection was performed with peroxide solution and luminol enhancer solution
- due to the anti-his tag antibody the presence of the protein is varyfied
- However there is no specific interaction between the anti-clostridium tetani antibody and the antigen
- for this experiment only the supernatant after sonification was used, not the purified protein
pET_1003; pET_1703
In further experiments we could not observe any expression of the target protein Hepatitis C Core Protein (pET_1003) and only less protein of HIV tat/poI/gag/env in the supernatant. Therefore, we tried another expression E.coli strain Bl21pLys and test several different temperatures and IPTG concentrations.
pET_1003
- in C43 strain, conditions: 37°C, 0.5 mM IPTG, 4h; 30°C, 1mM IPTG, o/n
- in Bl21pLys, conditions: 28°C, 1mM IPTG, o/n; 18°C, 1mM IPTG, o/n
pET_1703
- in Bl21pLys, conditions: 28°C, 0.1mM + 1mM IPTG, o/n; 18°C, 0.1mM + 1mM IPTG, o/n
13.07.2015
- o/n cultures pET_1103 (C42 + Bl21pLys), pET_1703 (Bl21pLys)
pET_1103
- inoculate 500ml LB-medium with 5 ml o/n culture [Amp+Cml], cells were grown until OD600 ~ 0.5
- induction with o.5 mM IPTG and grown 4h, 37°C
- cells were harvested by centrifugation 5000g, 25min, 4°C
- and frozen at -20°C for the protein purification
12.07.2015
- transformation of pET_1103 und pET_1703 in C43 and Bl21pLys
- o/n culture of glycerol stock pET_1103 C43
10.07.2015
pET_1103; pET_1003; pET_1703 C43
- 12,5% SDS-PAGE gel with all conditions for western blotting
- Semi-dry blotting was performed with PVDF membrane (1h, 0.1A)
- the membrane was washed twice for 10min in TBS buffer at RT
- Incubation for 1h in blocking solution at RT (special blocking solution of Qiagen; 1xblocking buffer, 0.5% blocking powder, 0.1% Tween)
- afterwards membrane was washed for 10 min in TBS-Tween at RT
- Following the incubation in Anti-His HRP Conjugate (1:1000 in blocking buffer)
- membrane was washed twice for 10 min in TBS-T
- Chemiluminescent detection was performed with peroxide solution and luminol enhancer solution
Result
- Atfer the detection membrane was stained with coomassie. The samples of pET_1003 showed clear stained proteins. The western blotting confirmed no his-tagged protein in this samples
- pET_1103 shows also in the supernatant the target protein, whether the samples of pET_1703 indicates the protein in the pellet
09.07.2015
pET_1103; pET_1003; pET_1703 C43
- sonification of 37°C (0.5 mM IPTG) and 30°C (1 mM IPTG) samples [6cycles, 5min, 75% intensity]
- addition of PMSF 1:1000 in lysis buffer (stock 300mM)
- centrifugation 10 000xg, 30min, 4°C
- 12,5% SDS-PAGE Analysis to verify, whether the protein is in the supernatant or in the pellet (inclusion bodies)
Results
- pET_1003: Hepatitis C Core Protein = 21 kDa
- pET_1103: Clostridium tetani TeNT_Hc = 50 kDa
- pET_1703: HIV tat/poI/gag/env = 20 kDa
for pET_1003 and pET_1103 no obvious enchrichment of the target protein. For pET_1703 the protein seems to be in the pellet. For further analysis a western blot with anti-His Tag antibody is going to be performed with all samples
08.07.2015
pET_1103 C43
Clostridium tetani TeNT_Hc antigen overexpression test in C43
- inoculate 30ml and 50ml LB-medium [Amp+Cml] with the o/n culture, respectively.
- glyercol stock in -80°C
pET_1003 C43
Hepatitis C Core Protein antigen overexpression test in C43
- inoculate 30ml and 50ml LB-medium [Amp+Cml] with the o/n culture, respectively.
- glyercol stock in -80°C
pET_1703 C43
HIV antigen overexpression test in C43
- inoculate 30ml and 50ml LB-medium [Amp+Cml] with the o/n culture, respectively.
- glyercol stock in -80°C
pET_803 Bl21pLys
for overexpression pET_803 (Herpes simplex type 1 glycoprotein 1) and protein purification. the pre-overexpression test was performed 4./5.7
- inoculate 500ml LB-medium [Amp+Cml] with the o/n culture
- glyercol stock in -80°C
07.07.2015
o/n culture [Amp+Cml] pET_803;1003;1103;1703 (6ml)
06.07.2015
pET_1703
transformation of plasmid in E.coli overexpression strain C43 protocol transformation Resistance: plasmid (Amp); strain (Cml)
pET_1103
transformation of plasmid in E.coli overexpression strain C43 protocol transformation Resistance: plasmid (Amp); strain (Cml)
pET_1003
transformation of plasmid in E.coli overexpression strain C43 protocol transformation Resistance: plasmid (Amp); strain (Cml)
05.07.2015
SpyCatcher C43
- Gels were destained o/n and photos taken
Results
- Spy Catcher expression seems not to have been succesfull
- Retry with a different expression strain or a fusion protein
- fusion to mCherry would allow for direct coloclisation experiments with SpyTag-labeled GFP-protein
pET_803 overexpression in BL21
- o/n stored pellets were resuspended in 5 mL Lysis Buffer each by 20 sec sonification
- stored @ 4°C
- samples from supernatant and pellet (9 µL + 6 µL 2.5x SDS-buffer) were boiled on 95°C for 5 min
- store @ RT for gel loading
Results: Whole cell lysate
- Results are promising that overexpresison was succesfull
- Gels from sonification have to show if the protein in soluble
Results: Sonification
- Sonification shows the antigen as soluble and most expressed at 37°C 4 hours incubation in BL21
- The overnight condition cannot be evaluated as staining was not very succesfull
- If purification from the bigger batch does not work: keep in mind that overexpression conditions were not tested with OD_600 = 0.5 as starting conditition
04.07.2015
cell disruption pET15_803, SDS-PAGE 12%
- 1 ml and 10 ml samples of overnight cultures of induced cells (pIG15_803) were taken
- 1 ml samples were prepared for SDS-PAGE as day before (centrifuge, resuspend the pellet in 2.5xSDS buffer and boil for 10 min.)
- perforemed 12% SDS-PAGE with 1 ml samples of BL21 before induction, 2h and 4h 18°, 28° and 37° each:
Gel usage
| 4µl ProSieve Quad Color Protein Marker | 12µl before induction BL21 | 12 µl BL21 2h 18° 0.1 mM IPTG | 10 µl BL21 2h 28° 0.1 mM IPTG (added 50 µl of 2.5xSDS buffer, because I was unable to pipet it first) | 15 µl BL21 2h 37° 0.1 mM IPTG | 12 µl BL21 2h 18° 1 mM IPTG | 12 µl BL21 2h 28° 1 mM IPTG | 12 µl BL21 2h 37° 1 mM IPTG | 12 µl BL21 4h 18° 0.1 mM IPTG | 12 µl BL21 4h 28° 0.1 mM IPTG | 12 µl BL21 4h 37° 0.1 mM IPTG | 12 µl BL21 4h 18° 1 mM IPTG | 12 µl BL21 4h 28° 1 mM IPTG | 12 µl BL21 4h 37° 1 mM IPTG | 4 µl ProSieve Quad Color Protein Marker |
- Commassie staining and destaining of gel
- sonification (60 %, Mode 6, 5 min) and 1:1000 300 mM PMSF in ethanol for 10 ml samples pIG15_803
- 30 min, 10000 g 4°C
- separeated supernatant and pellet (aliquot from supernatant)
- froze both at -20°C (in labelled primer box, freezer second drawer from above
03.07.2015
SpyCatcher in C43: harvest
- cells (32.5 mL) were harvested by centrifugation (5000g, 20min, 4°C)
- cells from this harvest and from yesterdays were resuspended in 7.5 mL lysis buffer each
- sonification (60 %, Mode 6, 5 min) and 1:1000 300 mM PMSF in ethanol
- 30 min, 10000 g 4°C
- separeated supernatant and pellet (aliquot from supernatant)
- resuspended pellet in 7.5 mL Lysis buffer (1 min sonification)
- suspended 9 µL of each pellet or supernatant in 6 µL 2.5x SDS-buffer
- 5 min 95°c
- SDS-PAGE (15% Gel)
Gel usage
| x | x | 37°C 0.5 mM Supernatant | 37°C 0.5 mM Pellet | 30°C 1 mM Supernatant | 30 °C 1 mM Pellet | ProSieve Quad Color Protein Marker | before induction | x | x | x | x | x | x | x |
37°C Pellet was not pipettable and used 1:2 diluted in 2.5x loading buffer
- on first look there seem to be no bands with a molecular weight lower than 25 kDa
o/n culture of C43 SpyCatcher was canceled as there seems to be no expression
DotBlot: antibodies
- antibody solution goat anti-GFP (the one the iRIf group got from Max) 1:1000 in PBS 5% milk was prepared
- one membrane was incubated with this solution
- the other membrane was incubated with rabiit anti-GFP 1:1000, 0.05 % Sodium azide (from the cellfree expresison group)
- incubation for 2.5 h
- incubation in TBS-T over day
- anti-goat HRP couldn't be found → membrane was further incubated in TBS-T
- mouse anti-GFP membrane was incubated 1 h in anti-mouse HRP (5 % milk powder in PBS)
- detection in ECL reader
Results
- The mouse anti-GFP binding is strongest to the purified GFP from AG Ublrich even though all samples were normalized on fluorescence and should therefore contain comparable amounts of fluorophors
- In the mCherry control no binding is visible → low amount of unspecific binding
- The binding intensity for the two AG Weber samples is equal as expected from the same sample stored in different concentrations
pET_803: Overexpression test in BL21 and C43
- for pre-culture of BL21 cells
- 3 200 mL and 3 500 mL flasks were filled with 100 mL or 200 mL LB-medium respectively
- 1:1000 Amp was added (100µL and 200 µL respectively)
- cultures were started with 0.5 or 1 mL o/n culture respectively (only 5 mL culture in toto)
- incubation @ 37°C
- for pre-culture of C43-cells
- 2 50 mL flasks were filled with 25 mL LB-medium
- 1:1000 Amp was added (25 µL)
- cultures were started with 25 µL o/n culture
- incubation @ 37°C
- cultures were around OD_600 0.2 after 1.5 h
- cultures were grown to OD_600 of 2 after 2.5 h
- diluted cultures with LB-Medium to an OD_600 of 0.5
- added Amp (relative to the dilution volume)
- incubation 0.5 h at 37°C 200 rpm
- took aliquots (1 mL) from BL21 and C43 cells
- induction with 0.1, 0.5 or 1 mM IPTG
- OD_600 was again between 1 an 1.5 for the BL21 cultures and around 1.6 for C43
- spin down aliquots 15000 g 2 min → resuspended in 200 µL 2.5x SDS-loading buffer
Cells are in exponential growth but may already be too dense for induction
- after 2h 37°C 0.5 mM IPTG C43 culture was at OD_600 of 4.5 and BL21 28°C 1mM IPTG culture was at 3.84 → cells seem to grow
- 10 mL for sonification and 1 mL for the gel were taken 2h and 4h after induction
02.07.2015
pET_Spy catcher C43
In this experiment we try to overexpress and purify the spy catcher (2kDa + 1kDa His-Tag) by Ni-NTA
- o/n culture on plate for further experiments
- inoculate 2x30ml samples for overexpression test
- cells are grown until OD600 ~0.5 and induced with IPTG 0.5mM (37°C,4h) and 1mM (30°C, o/n), respectively.
- 4h after induction the 37°C sample was harvested by centrifugation (5000xg, 20min, 4°C), pellet in -20°C freezer
pET_803 (Herpes simplex) C43 + Bl21pLys
- o/n culutres for overexpression test on 03.07
- transformed pET_803 in C43 and Bl21pLys for comparison of overexpression results of pIG15_803 (own overexpression backbone. overexpression with this antigen failed in our own backbone)
Mini-prep for constructs interlab study
Mini-prep was performed with the Qiagen Kit
Concentrations:
Bba_823005 -1 46,1 ng/ul
Bba_ 823008 -1 67,9 ng/ul
Bba_ I13504 -2 101,5 ng/ul
Bba_ I13504 -1 66,8 ng/ul
Bba_8230/3 - 2 72,7 ng/ul
Bba_ 8230/3 -1 113,9 ng/ul
→ in der Box tagged-proteins damit du sie findest; o/n culture für mini prep. hab ich dir auf Platte (Cml), ist im 37°C
Western blot, pIG15_1301, pIG15_1501
The western blot should confirm, whether the purified proteins are enriched. For the specific detection anti-his-tag antibody is used. All purified proteins are his-tagged. We used the monoclonal anti-his conjugated antibody from Qiagen. Purified GFP-His and crude lysate with overexpressed GFP-his were used for positiv control.
- 12% SDS-PAGE gel was used for the separation of the proteins according to their molecular weight
- Semi-dry blotting was performed with PVDF membrane (1h, 0.1A)
- afterwards the membrane was stained with ponceau solution to check, if the transfer was efficient.
- the membrane was washed twice for 10min in TBS buffer at RT
- Incubation for 1h in blocking solution at RT (special blocking solution of Qiagen; 1xblocking buffer, 0.5% blocking powder, 0.1% Tween)
- afterwards membrane was washed for 10 min in TBS-Tween at RT and 10 min in TBS buffer
- Following the incubation in Anti-His HRP Conjugate (1:1000 in blocking buffer)
- membrane was washed twice for 10 min in TBS-T and 10 min in TBS buffer
- Chemiluminescent detection was performed with peroxide solution and luminol enhancer solution
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WB confirms the coomassie gels of the protein purification. Additionally pIG15_1301 shows a molecular weight of 35kDa.
This leads to the assumption, that gIII is cleaved off after the transfer into the periplasm.
GFP-DotBlot
- Spotted 2 µL GFP on activated PVDF membranes in the following pattern
| GFP nT | GFP His | GFP Max |
| Weber GFP 8 mg/mL | Weber GFP 600 mg/mL | mCherry-His |
* blocked 10 min with 5 % milk in TBS-T
- washed 2x 10 min in TBS-T
- incubated o/n in anti-GFP monoclonal mouse (Phillip) or anti-GFP polyclonal rabbit (Max)
01.07.2015
pET_803 (Herpes simplex) C43 + Bl21pLys
- transformation of the plasmid pET_803 in C43 + Bl21pLys (this antigen we were not able to overexpress in our own expression backbone)
pET_Spy catcher C43
- o/n culture for overexpression test (Amp+Cml) [5ml]
