Immunoblotting using precast gels

Mix and in a 7:3 ratio (for example: 70µL LDS sample buffer and 30µL of Reducing Agent).

In a 1.5 mL microcentrifuge tube add the following reagents in order: LDS sample buffer/Reducing Agent mix, IPMS lysis buffer, protein sample. For example.

A	B
10 or 15 microliters	5 microliters
20	7
30	10

Poke hole in top of 1.5 mL microcentrifuge tube with a hypodermic needle.

Boil samples at 95°C for 0h 5m 0s using a ThermoMixer F1.5.

Equipment

Value	Label
Eppendorf ThermoMixer F1.5	NAME
ThermoMixer	TYPE
Eppendorf	BRAND
5384000020	SKU

Centrifuge for 0h 0m 10s in a Mini Centrifuge at Room temperature

Equipment

Value	Label
Fisherbrand Mini-Centrifuge	NAME
Centrifuge	TYPE
Fisherbrand	BRAND
12-006-901	SKU

Samples can be stored at -20°C until the day of running SDS-PAGE electrophoresis.

Prepare 500mL 1X MES-SDS Buffer (50millimolar (mM) MES, 50millimolar (mM) Tris Base, 0.1Mass / % volume SDS, 1Mass / % volume EDTA, pH 7.3) by diluting stock 1:20 in Milli-Q water.

Unpack a and remove protective tape and comb.

Assemble gel running tank by placing gel in the tank in front of the buffer core and a second gel or a buffer dam behind the

buffer core. Lock the gel tension wedge in place. Add 1X MES-SDS buffer between the gels and outside the buffer core.

Load 8µL protein molecular weight marker . If needed load 2µL at the end of the gel.

Load samples to a maximum volume of 20µL.

Place gel tank lid and connect to power supply matching colors (for positive and negative).

Run gel at 150 V for 2h 0m 0s at Room temperature or as needed depending on the protein molecular weight and desired separation.

Prepare 12X transfer buffer:

• 58 g Tris base
• 190 glycine
• Milli-Q water up to 2L

Prepare 2.5 L of 1X transfer buffer (48millimolar (mM) Tris, 39millimolar (mM) Glycine, 20% (v/v) Methanol) by mixing 212mL 12X Transfer buffer with 1788mL milli-Q water and 500mL Methanol.

Cut or membrane to the required gel size (usually 9x7 cm) and activate PVDF membrane by incubating in methanol for at least 0h 1m 0s.

Cut to the required gel size (usually 9x7 cm).

Prepare . If dirty, boil in warm tap water in the microwave for 0h 5m 0s. Soak sponge pads in 1X transfer buffer in a plastic tray.

Remove the gel from the cassette by separating the cassette plates with a . Using the gel knife, cut off the bottom part and wells of the gel as needed.

Place gel in a plastic tray with 1X transfer buffer.

Make transfer sandwich inside the cathode core of the blot module in the following order:

• 2 sponge Pad
• 1 filter paper + gel (grab this from transfer buffer tray), Remove air bubbles by carefully rolling a on top of the gel.
• 1 presoaked PVDF or Nitrocellulose membrane. Remove air bubbles using the Blot Roller.
• 1 filter paper. Remove air bubbles using the Blot Roller.
• Sponges up to fill cavity (6-7 total)

Close blot module with the anode core and place this in the buffer chamber. Lock the blot module using the gel tension wedge.

Place lid and connect to power supply matching colors (for positive and negative).

Run gel at 35 V for 1h 30m 0s at Room temperature .

Unlock blot module by releasing the tension wedge and remove from buffer chamber. Remove membrane from the sandwich and cut excess membrane using scissors.

Incubate membrane in in a plastic tray for 0h 5m 0s with shaking in a rocking shaker.

Equipment

Value	Label
Rocking Shaker	NAME
Shaker	TYPE
Ohaus	BRAND
30391966	SKU

Wash with milli-Q water 3 times, quick washes. Image in Chemidoc MP if needed.

Equipment

Value	Label
ChemiDoc™ MP Imaging System	NAME
Imaging System	TYPE
Bio-rad	BRAND
12003154	SKU
http://www.bio-rad.com/	LINK

Make 1X TBS-T (Tris buffer saline-Tween 20: 20millimolar (mM) Tris-HCl pH=7.4, 100millimolar (mM) NaCl, 0.1% (v/v) Tween-20).

For 1L:

• 20mL 1Molarity (M) Tris pH=7.4
• 20mL 5Molarity (M) NaCl
• 1mL Tween-20
• Milli-Q water up to 1L

Make Blocking solution: 1X TBS-T 5% skimmed milk:

• 12.5g powder skimmed milk .
• Fill up to 250mL with 1X TBS-T.

Add Blocking solution to a Western Blot box and place the membrane in this solution. Incubate in a rocking shaker for at least 1h 0m 0s at Room temperature

Prepare antibody in 1X blocking solution (for HRP detection) or in TBST 5Mass / % volume BSA (for infrared detection) at the desired concentration.

Discard blocking solution from Western blot box and replace by diluted antibody. Incubate at 4°C 1h 0m 0s in rocking shaker.

Next day recover antibody in tube and wash membrane 3 times for 0h 5m 0s with 1X TBS-T

Prepare HRP or fluorescently labelled secondary antibody in blocking solution or TBST respectively.

Add diluted secondary antibody to Western blot box a incubate at Room temperature for 1h 0m 0s in rocking shaker.

Wash membrane 4 times for 0h 5m 0s with 1X TBS-T

Image in Chemidoc MP for HRP detection or LI-COR Odyssey FC imager for infrared detection.

Equipment

Value	Label
ChemiDoc™ MP Imaging System	NAME
Imaging System	TYPE
Bio-rad	BRAND
12003154	SKU
http://www.bio-rad.com/	LINK

Equipment

Value	Label
Odyssey CLx	NAME
Imaging System	TYPE
LI-COR	BRAND
Odyssey CLx	SKU

Membrane can be stored up to a week at 4°C until performing stripping.

Prepare Stripping buffer:

• weight 15g glycine
• Dissolve in 800mL milli-Q water
• Adjust pH to 2.2
• add 1g SDS
• 10 mL Tween 20
• Bring volume up to 1 L with milli-Q water

Incubate membrane 0h 10m 0s with stripping buffer in rocking shaker at Room temperature.

Repeat step 37

Wash twice with TBST

and probe with a different antibody.