Team:Heidelberg/notebook/ab
week number 37
▼2015-09-10 E. coli llysate
inoculate overnight culture of E. coli
next day delute culture 1:50
incubate culture at 37°C till OD600=0.8
add 1mM IPTG
let E. coli grow for 2h
take 1.5ml E.coli
centrifuge for 2min full speed
remove supernatant
resuspent pellet in 500µl lysis buffer with protease inhibitor
sonication 30sec; 50% power 3x
add DNase, RNase, both or no
let it incubate for 30min
add laemmli buffer heat up for 5min at 95°C
run SDS-PAGE
perform Western Blot according to the protocol
week number 35
▼2015-08-30 General Antibody Protocol
Western Blot
Cut pvdf membrane (9x6cm) and Blotting paper to the dimensions of the gel. One membrane and two pieces of extra thick filter paper per gel are needed
Activate membrane 5min in Methanol
Equilibrate gel in transfer buffer for 5min
Assembly:
1. Place a pre-soaked sheet of extra thick filter paper onto the platinum anode. Roll a pipet or test tube over the surface of the filter paper (like a rolling pin) to exclude all air bubbles.
2. Carefully place the equilibrated gel on top of the transfer membrane, aligning the gel on the center of the membrane. Transfer will be incomplete if any portion of the gel is outside the blotting media. Roll out all air bubbles.
3. Place the other sheet of pre-soaked filter paper on top of the gel, carefully removing air bubbles from between the gel and filter paper.
4. run Blot at 25 V for 1h
Prepare around 50ml 5% milk in TBT-T.
After blotting wash membrane three times with TBT-T for 5min
Block 1h or overnight with 5% milk, keep milk afterwards
Wash membrane three times, 5min with TBT-T
Prepare dilution of first antibody in 5 ml milk in a Falcon
Put membrane in the Falcon and let it roll at 4°C overnight
Wash membrane three times, 5min with TBT-T
Prepare dilution of second antibody in 5 ml milk in a Falcon
Put membrane in the Falcon and let it roll at 4°C for 1 h
Wash membrane three times, 5min with TBT-T
Prepare strain solution: 2.5 ml each in one Falcon
Put membrane in the Falcon and let it roll at 4°C for 5min
Pipet 50 µl H20 on a clear film, add membrane, pipet 50µl on the membrane and close clear film
▼2015-08-30 General AptaBody protocol
AptaBody Western Blot
1. Run SDS-PAGE and do Western Bot transfer following common protocols
2.Prepare 5% milk or BSA in TBS-T.
3. After blotting wash membrane three times, 5min with TBS-T
4. Block 1h with 5% milk in TBST
NOTE: For very fast results you can skip the blocking step, however background may the higher
5. After blocking wash membrane three times, 5min with TBS-T
6. Prepare Anti-His-tag reaction solution:
• Fill up “His-Tag AptaBody” with 50 µl H2O
• Heat up “His-Tag AptaBody” at 95°C for 5min
• Fill up “Catalyst for AptaBody” with 50 µl H2O
• Mix equal volumes of both solutions
• Let reaction solution stand for 10min
7. Add 15µl Anti-His-tag reaction solution to 5 ml TBS-T in a Falcon.
8. Put membrane in the Falcon and let it roll at room temperature for at least 1h.
9. Wash membrane three times, 5min with TBT-T
10. Prepare ECL Western blotting substrate according to manufactural protocol.
11. Pipet 50 µl H20 on a clear film, add membrane, pipet 50 µl H20 on the membrane and close clear film
12. detect chemiluminescence with the proper imaging system.