Schedule, Notes; tips and tricks

Schedule

1st day

• Sample preparation (can be mixed with lämmli; store in -20°C
• gel casting (2h)
  

2nd day

• SDS-page (1 – 2 h), transfer (2 h), blocking (1h) primary antibody (O/N)
  

3rd day

• Secondary antibody (2h), acquisition

Prior start

check if you have propriate reagent/inhibitors phosphorylation: aware the phosphorylation sites prepare buffers etc.

if your protein tends to create condensates (liquid phase separation) – you can consider prolonged time of boiling (for 5 min to 30 min) some proteins should be heated up to lower temperate (such as Brca2 only 60°C)

Gel casting

• necessities: device for gel casting (casting stand, green casting frames); glasses (1.5 mm or 1 mm); propriate comb thickness (1.5 mm or 1 mm); reagent for gel
• clean glasses with EtOH before use
• Optional (not recommended): A) You can use parafilm to seal the bottom or B) prepare a little bit of gel with bigger amount of APS and TEMED – polymerize quickly – and pour it fist (a few millimeters) - to seal the bottom; with biorad it is not necessary – it usually fine
• Workflow: prepare (10 mL/1 gel) and pour resolving gel (keep space for comb + a little bit for stacking gel) → overlay with isopropanol (possible to use also H2O) → wait for polymerization (keep a little bit o gel in falcon and check progress there); approx. 30 min → dispose isopropanol → wash gel with H2O and dry leftover liquid with blotting/filtrate (wattman) paper; gently, do not touch the gel → prepare and pour stacking gel → place a comb → wait for polymerization (keep a little bit o gel in falcon and check progress there); approx. 30 min
• Storage: use immediately; or store O/N wrapped casted gel in wet tissue (in plastic bag) at 4°C; prolong storage can influence resolving capacity of gel

Protein size	Gel procentage
4 – 40 kDa	Up to 20 %
12 – 45 kDa	15 %
10 – 70 kDa	12.5 %
15 – 100 kDa	10 %
50 – 200 kDa	8 %
> 200 kDa	4 – 6 %

ELFO

• necessities: device for electrophoresis; gel(s); if you have odd number of gel(s) – fake plastic glass if you have leakage of running buffer: assembly again (keep running buffer in clean container and use it again) or pour more running buffer to maintain balance (principle of communicating vases)
• 37 μL (40 μL) of total volume is max loading volume/well, gel 1 mm tick biorad gel
• If gel runs unequally – you can consider load only buffer with lämmli to empty wells next time … fill all wells do not leave gel w/o current for so long – the molecules can diffuse (Brown motion)

Transfer

• necessities: device for transfer; buffers (listed in transfer notes); MeOH – for activation PVDF membrane; blotting papers (12/one gel); membrane (propriate size; slightly bigger than membrane); tweezers; green (flat) gel spatula; optional: 15-mL falcon to get out bubbles
• cut membrane to fit size of gel

• try to touch membrane as less as possible, use tweezers

• Transferring membrane to falcons (antibodies) wet the walls of the falcon with solution to reduce a friction of the membrane

Be aware of post-transcription modification of proteins

Phosphatase inhibitor: !!! fresh protease and phosphatase inhibitor cocktails !!! !!! check storage condition and toxicity !!!

e.g. mTOR is phosphorylated on several residues, including T2446, S2448, and S2481. T2446 is phosphorylated in response to nutrient availability. Initially, S2448 was reported to be an Akt phosphorylation site because its phosphorylation is sensitive to PI3K inhibition, which reduces Akt activity.
note: threonine Thr T

Cmp	Mw	Target class	Solubility (H2O)	working conc.
NaF	42.0	Ser/Thr and acidic phosphatase	40 mg/mL	1 - 20 mM
NaVO4	183.9	Tyr and alkaline phosphatases	20 mg/mL	1 - 100 mM
PMSF	174.2	Serine proteases	18 mg/mL (MeOH)	0.1 - 1.0 mM

Resources

Thermo Fisher: Overview of Protease and Phosphatase Inhibition for Protein Preparation