Drosophila embryos – Protein sample preparation

1) Collect 50–100 dechorionated embryos and transfer into 50 µl ice-cold lysis buffer in a 1.5 mL microcentrifuge tube (lysis buffer: 50 mM Tris pH8, 150 mM NaCl, 0.5% Triton X-100, 1 mM MgCl₂, 0.1 mM EDTA and protease inhibitor (e.g. cOmplete^TM EDTA-free Protease inhibitor Cocktail (Roche)).

2) Centrifuge for 1 min (20.000g, RT).

3) Use a tightly fitting pestle to homogenize the embryos (~5-10 strokes).

4) Centrifuge for 1 min (20.000g, @4C).

5) Repeat step 3-4.

6) Transfer lysate into a QIAshredder Mini Spin Column (Qiagen).

7) Centrifuge for 2 min (20.000g, @4C) and transfer flow-through to a new 1.5 mL microcentrifuge tube.

Alternatively: Sonicate the sample using a Diagenode Bioruptor for 7min (30s interval, power setting: ‘low’). Centrifuge for 10 min (20.000g, @4C). Transfer supernatant to a new microcentrifuge tube.

Optional (but recommended): To estimate the range of protein concentration in the sample, use e.g. a Qubit fluorometer with a Qubit Protein Assay Kit (Invitrogen) following the manufacturer’s instructions; or alternatively a BCA assay.

8) Add 1 volume of 4x Laemmli buffer.

9) Heat the sample to 95C for 10 min (e.g. in a heat block).

10) The samples can now be loaded for SDS PAGE.

Optional: Samples can be stored at -80C.

Western Blot

Buffers for Gel electrophoresis and Transfer

10x Running

250 mM Tris, pH 8.3

1.92 M Glycine

1% SDS

For 1 Liter:

30.4 g Tris

144 g Glycine

10g SDS

10x Transfer

(w/o MeOH)

250 mM Tris, pH 8.3

1.92 M Glycine

For 1 Liter:

30.4 g Tris

144 g Glycine

1x Transfer

(with MeOH)

25 mM Tris, pH 8.3

192 mM Glycine

20% MeOH

For 1 Liter:

100 mL 10x Transfer buffer

200 mL MeOH

TBS-T

1X Tris-buffered saline

(20 mM Tris, pH approx. 7.4, and 150mM NaCl)

0.1% Tween-20

Blocking solution

TBS-T

5% milk powder

For 100 mL:

100 mL TBS-T

5 g milk powder

10x Ponceau stain

30% (w/v) Trichloroacetic acid (TCA)

2% (w/v) PonceauS powder

For 100 mL:

30g TCA

2g PonceauS powder

Other Equipment/Consumables

Primary antibody for protein of interest; secondary antibodies: HRP-conjugated (for chemiluminescence); fluorophore-conjugated (for fluorescence)
Electrophoresis system (We used: Biorad Mini Protean system)
Transfer apparatus incl. foam pads and frozen cooling unit (We used: Biorad Mini Trans-Blot Electrophoretic Transfer Cell)
Polyacrylamid-gel (We used: BioRad 4–20% Mini-PROTEAN TGX gradient gels)
2 sheets of Whatman paper (9 x 9 cm)
1 sheet nitrocellulose or PVDF membrane (8.5 x 6 cm) (We used PVDF)
Cling film
Tweezers for handling membrane
Western Blot roller or 10 ml pipette
For chemiluminescence: we used Pierce ECL Western Blotting Substrate (Thermo Fisher)
Optional: Sturdy transparent plastic sheets (like the ones for old overhead projectors)
Optional: X-Ray film cassette and X-Ray film (e.g. Fuji RX-N 18x24cm)

Gel Electrophoresis (SDS PAGE)

1) Mount a gel in the electrophoresis apparatus. We used the Biorad Mini Protean electrophoresis system with BioRad 4–20% Mini-PROTEAN TGX gradient gels. Fill the chambers with 1x Running buffer.

2) Heat the sample to 95C for 10 min (e.g. in a heat block) before loading.

3) Load molecular weight marker and protein extracts (recommended: 5-50µg of total protein) into wells of the SDS polyacrylamide gel.

4) Run gel at 120V for ~1h.

Transfer

Note: Use FL-Immobilon PVDF membrane when using fluorescent secondary antibodies.

1) If using PVDF membranes for transfer, activate for 5 min in MeOH, rinse with water and place in transfer buffer.

2) Pre-wet foam pads, Whatman paper, membrane in cold transfer buffer.

3) Remove gel from running apparatus, cut off stacking gel.

4) Preincubate gel for some minutes in Transfer buffer.

5) Assemble the Western Blot in a cassette of a Mini-Protean Wet blot electrophoresis tank as follows:

Cathode (+, red/transparent)

Foam pad

Whatman paper

PVDF membrane

Gel

Whatman paper

Foam pad

Anode (-, black/black)

CRITICAL STEP: Ensure to remove air bubbles between the gel and the membrane as these would prevent the transfer of proteins.

6) Insert the assembled sandwich into a blotting chamber with a cooling aggregate and Transfer buffer.

7) CRITICAL STEP: Make sure that the membrane is facing the cathode (red) to allow the transfer of proteins from the gel onto the membrane. Ensure that the Transfer buffer covers the cassette. Run transfer at 100 V for 1-1.5 hours.

8) Disassemble apparatus, move membrane into clean gel dish filled with Ponceau stain and incubate for ~2 min.

9) Tip off Ponceau stain (back into bottle for re-use) and rinse gel with deionised water until bands can be seen clearly.

10) Tip off water and quickly take photo of membrane.

Optional: Cut membrane as appropriate.

11) Add TBS-T and place gel on rocker for 10 min to wash away remaining Ponceau stain.

Antibody incubation

1) Tip off TBS-T buffer.

2) Add blocking solution and place on rocker @ RT for at least 1h to block.

3) Tip off buffer.

4) Add primary antibody (Ab) in blocking solution (using as little as possible to cover gel) and incubate on a rocker overnight @4C.

5) 3 x wash membrane with TBS-T or blocking solution for 5-10 min on rocker @ RT.

6) Add secondary Ab (HRP-conjugated for chemiluminescence or fluorophore-conjugated for fluorescence) in blocking solution and incubate on a rocker for 1h @RT covered with aluminium foil.

7) 3x wash membrane with TBS-T for 5-10 min on rocker @ RT covered with aluminium foil.

8) Rinse membrane briefly in TBS (no Tween).

For fluorescence (if using fluorophore-conjugated secondary Abs):

9) Place membrane into LI-COR Odyssey M or DLx imaging system (or equivalent) and image.

For chemiluminescence (if using HRP-conjugated secondary Ab):

9) Prepare ECL Reagent (e.g. Pierce ECL Western Blotting Substrate (Thermo Fisher)) according to the manufacturer’s instructions (equal amounts of solution A and B).

10) Incubate the membrane with the ECL solution for 3 min.

11) Remove excess moisture from membrane by blotting back side of membrane on Kimwipes.

12) Place the membrane between two plastic sheets (or cling film).

13) If you have access to a Biorad ChemiDoc imaging system (or equivalent), place the wrapped membrane into the unit and image.

Alternatively:

13) Stick the wrapped membrane on a bit of Whatman paper and place into an X-ray film cassette.

14) In a dark room, mark a corner of an X-ray film, then quickly lay onto blot and close film cassette (leaving for 1 min to start; then try longer exposure times).

15) Quickly remove film and place into the developer long-edge first, aligned to the left-hand side of the input tray.