Team:Freiburg/Labjournals/ProtPur/August
Protein Purification
21.08.2015
Salmonella Antigen (dehydroxy acid dehydratase) and anti-dehydroxy acid dehydratase
GFP
17.08.2015
Salmonella Antigen (dehydroxy acid dehydratase) and anti-dehydroxy acid dehydratase
Given that we could measure a binding of pIG15_1301 to pIG15_1501, we wanted to proof this binding event by western blot. The primary antibody pIG15_1301 (anti-dehydroxy acid dehydratase) is selfmade by overexpression in E.coli and purified with Ni-NTA. These single chain antibodies contain beside the his-tag for purification an additional cmyc-tag. The antigen (pIG15_1501) does not contain any cmyc-tag. Therefore we used as secondary antibody anti-cmyc (rabbit). For the readout we are using chemilumineszenz immunoassay. So we used as third antibody the anti-rabbit Horseradish Peroxidase.
In addition to this western blot we also performed a western blot against the anit-his-Tag. There we determined the presence of our purified proteins, which are containing the his-tag.
The concentrations of the antibody were used in the following dilutions:
- anti-his conjugated (1:1000)
- anti-pIG15_1501 (1301) 1:100
- anti-cmyc 1:1000
- anti-rabbit HRP 1:5000
HA-tag-GFP-His-tag
As positive control in our measurement device, we are using GFP-His-Tag and anti-GFP biotinylated. This binding event we determined by western blot.
14.08.2015
purification of pET15_1003;1103;1703 and pIG15_1301;1501
Results
pET15_1003 protein purfication
pET_1103 protein purification
pIG15_1301 and pIG15_1501 protein purification
pIG15_1501 and pET15_1703 protein purification
- Flow-through of the desalting step indicates no loss of protein during this procedure.
13.08.2015
purification of pET15_1003;1103;1703 and pIG15_1301;1501
- the purification was performed according to Protein Purification on gravity flow columns
- all steps were performed in the cold room (4°C) and no vacuum was used.
- after the elution step, the samples were combined to 2,5 ml and desalted with specific columns for buffer exchange.
- the final concentration of the buffer is 20mM Tris, 150mM NaCl and 30mM Imidazole
- afterwards the protein concentration of the samples was determined
10-12.08.2015
- Interlabstudy
09.08.2015
overexpression test SpyCatcher
Results
Sequence of SpyCatcher was incorrectly annotated. We did not overepxress the SpyCatcher
08.08.2015
pET_Spy Bl1pLys
- 100ml LB (Amp+Cml) was inoculated with 1ml o/n culture
- grow until OD600~0.5 and induced with 1mM IPTG
- cells grow for 3h hours at 37°C
- cell harvest and lysis after protocol
- samples: 10' after induction, supernatant and pellet of the lysis → overexpression should be visible on the gel
- 15 mL LB (+Amp + Cml) was inoculated with pET_Spy BL21 pLys and grown o/n
Results
The expected molecular weight of the SpyCatcher-GST-complex is around 32 kDa and thus may fit to the strong band in the pellet fraction gel lane.
SpyCatcher-GST protein band is only visible for the pellet fraction.
To confirm the results a second SDS-PAGE was performed, starting from the frozen lysate and pellet resuspension. This experiment confirms the first data that the SpyCatcher-GST fusion protein ist located in the pellet.
The used fusion protein seems to be not GST-SpyCatcher but GST-SpyTag. That accounts for the lower molecular weight. Test for solubility has to be repeated with new GST-SpyCatcher construct.
07.08.2015
Western Blot phyA-Nterminal fragment
- a new Western Blot was performed with a 1:100 dilution of the purified phyA N-terminal fragment.
- The primary antibodies anti-phyA Agrisera and anti-phyA-Nterm were used 1:1000. The secondary antibody was diluted 1:5000 (anti-rabbit)
- The experiment was performed according to the protocol Western Blot towbin et al.
Results
mCherry-Halo; GFP-Halo; phyA
- all 500ml cultures were harvested by centrifugation 20 min, 5000g, 4°c
- each pellet was frozen at -20°C for further experiments
Arctic Express
- new competent cells were made of the E.coli strain Arctic Express (received of AG Weber)
- the protocol competent cells was used
06.08.2015
pET_SpyC_GST
GFP_Halo, mCherry_Halo
- inoculated 500 ml Amp/Cml with o/n culture
- growth at 37°C
- induced with 1 mM IPTG at OD ~ 0.45
- growth o/n at 28°C
phyA_ArcticExpress
- inoculated 500 ml Amp/gent with o/n culture
- 3 h growth at 37°C
- 1 mM IPTG induction
- growth o/n at 10°C
05.08.2015
pET_SpyC_GST
- Transformation in E. coli BL21 pLys (Amp/Cml)
GFP_Halo, mCherry_Halo
- inoculated 5 ml o/n culture
phyA_ArcticExpress
- inoculated 100 ml o/n culture
04.08.2015
new western blots of pET_1003; pET_1703; phyA with lower antibody and protein concentration
Western Blotting was performed according to the Western Blot towbin et al. protocol.
pET_1003; pET_1103; pET_1703 anti-his conjugated
- Aim of this experiment was to determine the overexpressed his-tagged target protein. Therefore we used the special Qiagen Kit with the anti-his conjugated antibody.
pET_1003 anti-Heptatitis C Core Protein antibody polyclonal
(developed this membrane alone, to get a higher signal with a longer exposure time (2,40min).)
- In this experiment we wanted to show the specific binding of the polyclonal antibody to our target protein with a molecular weight of 22kDa
- primary antibody: Rabbit Anti-HCV Polyclonal 1:5000
- secondary antibody: anti-rabbit HRP 1:5000
western blot of anit-phyA and pET_1003; pET_1103; pET_1703
- In this experiment we wanted to show the specific binding of the polyclonal antibody to our target protein.
- pET_1003 (molecular weight 22kDa):
- primary antibody: Rabbit Anti-HCV Polyclonal 1:5000
- secondary antibody: anti-rabbit HRP 1:5000
- pET_1703 (molecular weight 20.5 kDa):
- primary antibody: Rabbit anti-HIV P24 Polyclonal Antibody 1:5000
- secondary antibody: anti-rabbit HRP 1:5000
- phyA (molecular weight 63 kDa):
- primary antibody: agrisera rabbit anti-phyA 1:1000; secondary: anti rabbit HRP 1:5000
- primary antibody: anti-Ath N-terminal fragment 1:1000; secondary: anti rabbit HRP 1:5000
02.08.2015
phyA
- purfication according to Protein Purification protocol
- purification was done without compressor
- whole protocol performed in 4°C room
- with the samples of the different purification steps a western blot for anti-his conjugatd as well as the specific anti-phyA antibody was performed Western Blot towbin et al.
| elution | concentration mg/ml |
|---|---|
| Elution 2 | 6.6 |
| Elution 3 | 2.3 |
Results
Western Blot of phyA
the white bands on the membrane are due to too much protein. These white ghosts bands are the result of quenchend ECL substrate. The western blot with the specific antibody will be repeated with less protein
12,5% SDS-PAGE Analysis of the purification
GFP/mCherry-Halo from AG Roth
- cells were harvested by centrifugation (3243 g 4°C 30min)
- pellet was resuspended in 50 mL Lysis buffer
- Centrifugation 20 min 3243 g 4°C
- pellet frozen @ -20°C
- the purification was peformed according the Cell disruption and Protein Purification on gravity flow columns protocols.
concentration by amido black
| protein | concentration mg/ml |
| GFP E1 | 8,1 |
| GFP E2 | 17,9 |
| GFP E3 | 1,1 |
| mCherry E1 | 11,1 |
| mCherry E2 | 21,6 |
| mcherry E3 | 1,1 |
Results
12,5% SDS-PAGE Analysis His-mCherry-Halo
12,5% SDS-PAGE Analysis His-GFP-Halo
Western Blot of His-mCherry-Halo and His-GFP-Halo
In this experiment we verified via western blotting using an anti-his conjugated antibody our his-tagged target proteins. Of both proteins too much protein was loaded. This resulted in quenched ECL substrate.
01.08.2015
GFP/mCherry-Halo from AG Roth
- day culture: 500 mL LB (+ amp) were inoculated with the o/n cultures
- at on OD600 of about 0.5 the colonies were induced by 1 mM IPTG
- growth o/n @ 28°C
phyA
- harvested cells after 23 h growth @ 10°C by centrifugation (20 min, 4°C 5000g)
- resuspend pellets in 50 mL Lysis buffer
- Centrifuged 20 min, 4°C 4320g
- stored @ -20°C
31.07.2015
GFP/mCherry-Halo from AG Roth
- got 2x 5mL inoculated LB-amp
- incubated 7h @ 37 °C
- inoculated a plate with 40 µL GFP-Halo and another with 20 µL mCherry-Halo
- prepared o/n cultures with 5 mL LB+amp and 200 µL mCHerry-Halo (as the cells were grown much faster) or 1 mL GFP-Halo
- incubated the cultures o/n @ 37 °C
Protocol/Information from Tobi (AG Roth)
- strain: BL21 (mCHerry-Halo) and BL21 pLys (GFP-Halo)
- Expression-protocol (was the only one Tobi has tested)
- Growth @ 37°C until OD600 = 0.4
- Induce with 1 mM IPTG
- Expression o/n @ 28°C
- vector maps were send to us by him
phyA purificaion
- 100 mL LB (amp + gen) was incoluated with the whole o/n culture
- growth 3h @ 37°C
- 450 mL LB (amp+gen) was inoculated with the 100 mL culture and grown @ 37°C for 3h
- culture was put @ 4°C for 30 min to cool down
- induction with 500 µL of 1M IPTG to achieve final concentration of around 1 mM
- growth for 24 h @ 10°C
purification of pET_1003; pET_1103; pET_1703 and pIG15_1301; pIG15_1501
- o/n grown samples of pET_1003, pE_T1703 & pIG15_1301 were harvested by centrifugation (20 min 5000g 4°C)
- pellets were resuspended in 50 mL Lysis buffer each
- second centrifugation (4320 g, 20min 4°C)
- supernatant was discarded & pellet frozen @ -20°C
