Sample preparation for Bis-Tris 4-12% gel (before start, preheat thermostat to 70C, at Thermomixer comfort-corner of bench) Take the appropriate amount of protein and add 4x(1:4)LDS, DTT (stock concentration is 1M, final concentration should be 50mM DTT, so it’s 0,5 ul of DTT in every 10 ul of loading volume).
you need to know the concentration of your samples and calculate your-own table depending on the concentration of protein you would use. Use the V=m/C (m is the amount of protein going to each well-constant-, C is the concentration of my protein sample)
–Also I recommend you to prepare Master Mix to avoid pipetting mistakes and reduce the time you spend to prepare samples. For example, you have 10 samples, so you would prepare a mix for 11 or even 12 samples because the pipetting process has some errors. Mix the DTT, 4xLDS and mQ (you will add mQ in the Master Mix if all of your samples have the same concentration otherwise you should add an appropriate volume of mQ to each sample). Since Master Mix is prepared you would use one clean tip to add the same amount of master mix to all clean eppendorfs. And then you will add samples and pipetting up and down to mix them well.
Then heat samples at 70C for 10’ (no shaking)
Spin max spin on top bench centrifuge to reduce bubbles
Remove the comb (gently) out from the cassette by using both thumbs, wash the wells with running buffer and dispose (2x), remove the tape at the bottom of casette, place the cassette according to instruction below. Use 1x MOPS and 350ul ANTIOXIDANT for small gel, double this in big gel.
Loading samples: always write the order before you load them to prevent confusion. Here is the video to remind you how to prepare tank for electrophoresis https://youtu.be/jQQ85_5CsdE
Don’t forget to load marker (usually 2nd well) ~ 5 ul (name of marker (our “standard” marker)
Load 1.1x LDS in empty wells (first and last) to run gel equable
The voltage and time would depend on which separation you want to reach. (e.g. start at 160 V until the samples reach the uppermost ledge(?), then raise the voltage to 200 V.
Use appropriate “SDS-PAGE”loading pipette tips The running buffer is non reusable, so bump it !! !! Wet transfer, 70V for 1.5hr
Transferring buffer reusable
Prapare 5% Milk (2,5 g for 50 ml of TBST buffer)
Blocking- Leave the membrane in milk for 1 hour- or overnight if its at cold-room (4 C)
Wash after milk with TBST (just quick wash)
Preparing primary and secondary Ab For Ab Dilution Buffer: TBSD, 5% BSA w/v, 0.2% NaN3 (preservative, !toxic!) Eg. 100ml TBSD, 5g DSA, 200ul NaN3
Washes (prefer the horizontal platform shaker) Anti-PRKACA (D38C6) 1st Ab - 1:1000 dilution - overnight in cold-room or 1 hour in RT. Shaking is required. Wash 10 min 3 times.
Change the plastic container (after 2nd wash) because primary antibodies stick to walls- wash the used one with detergent
Primary antibodies can be re-used up to ~6-7 times, so don’t dump them
Anti-rabbit 2nd Ab - 1:10.000 dilution (e.g. for 20ml of BSA/TBST/NaN3 use 2ul 2nd Ab) - 1 hour in RT. Shaking is required. Wash 10 min 3 times.
For membrane visualisation
-Spread HRP substrate (2-3ml) evenly around the membrane with pipette (cover whole membrane)
-Before you place it to the visualiser, make sure you drain the HRP substrate by sliding the membrane to the wall of container and wiping the membrane circumferentially with paper- also remove drops from membrane, if needed
Taking pictures of membrane
1 picture every 20-30 seconds while adjusting the parameters
