20200113: WB FLOT1 and patient’s sera

50 ug protein/well. We use 2 gels.

First Gel –> wells:

1. Marker
2. non-transfected HEK cells
3. FLOT1/2-transfected HEK cells
4. Rat brain
5. Rat cerebellum
6. Swine optic nerve
7. Mouse brain
8. SH-SY5Y (para comprovar que no tienen FLOT, porque las HEKs sin transfectar sí que tienen)
9. non-transfected HEK cells
10. FLOT1/2-transfected HEK cells

Second gel –> wells:

1. Marker
2. non-transfected HEK cells
3. FLOT1/2-transfected HEK cells
4. non-transfected HEK cells
5. FLOT1/2-transfected HEK cells
6. non-transfected HEK cells
7. FLOT1/2-transfected HEK cells
8. non-transfected HEK cells
9. FLOT1/2-transfected HEK cells
10. Marker

Protocol:

• We have used a precast gel (4-15% achrylamide) with 10 wells (Bio-Rad)
• Warm the samples at 96ºC for 5 minutes
• Load samples in the wells (30ul per well)
• Run with running buffer 1x (commercial –> BioRad) at 20 – 40mA
• Transfer to a nitrocellulose membrane with Transblot Turbo (using the commercial buffer) –> mixed proteins protocol
• Blotting: wash the membrane with water and cut it (in this case we have 6 membranes)
  • Blocking: blocking buffer Casein – PBS 1:1 1h
  • Primary antibodies (diluted in Casein-PBS 1:1) –> 2 hours at RT
    • Mb1 (from well 1 to 8) –> Ac anti-FLOT1 (rabbit) 1/100 + Ac anti-B actin (mouse) 1/20.000
    • Mb2  –> serum MS 158 1/50 + Ac anti-FLOT2 1/200
    • Mb3 –> serum MS 163 1/50 + Ac anti-FLOT2 1/200
    • Mb4  –> serum MS 247 1/50 + Ac anti-FLOT2 1/200
    • Mb5 –> serum MS 316 1/50 + Ac anti-FLOT2 1/200
    • Mb6 –> serum negative control 202-02 1/50 + Ac anti-FLOT2 1/200
  • Wash with PBS-tween0’1% (3×5′)
  • Secondary antibodies
    • Mb1: GAR800 + GAM680 1/7500 (diluted in Casein-PBS tween0’1%): 1h at RT
    • Mb2: GAH800 + GAR680 1/7500 (diluted in Casein-PBS tween0’1%): 1h at RT
  • Wash with PBS-tween0’1% 1x (2×5′)
  • Wash with PBS 1x (1×5′)
  • Read with Odyssey equipment

RESULT:

• Mb1: la banda de FLOT1 (verde) se ve en todos los carriles menos en el de SH-SY5Y. Se observa más FLOT1 en los tejidos (a diferencia de lo que veíamos con FLOT2). Los lisados son correctos (la B-actina se ve bien). En las HEKs no transfectadas también hay FLOT1 y FLOT2.
• Resto de membranas: hay mucho fondo rojo en los carriles con cels transfectadas FLOT1/2. Parece que se ve una banda en todos los pacientes que colocaliza con FLOT2, pero también parece que aparezca en el suero control negativo.