Novex Xcell II Mini-Cell Western Blot Protocol

Sample Preparation
Assembly
Loading
Gel Electrophoresis
Transfer/Blotting
Staining
Detection
 
 
 

Sample preparation


Thaw the samples on ice, and keep it on ice until samples are denatured to avoid degradation.
Use 25-100µg of protein and add lysis buffer up to 12,5µl.
Add 12.5µl of 2X Sample buffer.
Mix and incubate it at 85°C for 2 min to denature the proteins
.
Assembly

Loading


Load 20µl of sample per well
If all weels are not loaded with sample or marker, load the remaining wells with sample buffer

Gel Electrophoresis


Power supply settings: program 5; 125V, 40mA for 1.5h

Transfer/Blotting


Online manual pdf
  1. Cut nitrocellulose membrane in to size that covers the gel size
  2. Put membrane in transfer buffer for 5-10 minutes.
  3. Wet all except gel in transfer buffer; remove trapped air from sponges
  4. Make the sandwich  for transferring the proteins from the gel to the membrane; assemble in the following order:
UP DOWN -into the cathode core which is tray-shaped

Close the cassette and put in the apparatus. Pour off excess transfer buffer and keep the tray horizontal while securing it into the minicell apparatus to avoid leakage of transfer buffer into the main chamber (plastic is sensitive to methanol)

Perform transfer using power supply settings: program 6; 25V, 110mA for 1.5h
 
 

Staining

Put the membrane in PBS containing 0,5% Triton X-100, and 5% milk to block unspecific binding in shaker for 1-2 hours in room-temperature or overnight at 4°C

Wash the membrane 3x with PBS containing 0,5% Triton X-100.

Put 10 ml of PBS containing 0,5% Triton X-100, 5% milk and with primary antibody and incubate on a shaker for 1 hour in room temperature (incubation time depends on the antibody) or overnight at 4°C
 
 

Detection

Wash membrane 2x10 minutes with PBS containing 0,5% Triton X-100.

Put 10 ml of PBS containing 0,5% Triton X-100, 5% milk and with secondary antibody with HRP and incubate on a shaker for 1 hour in room temperature
 

  1. Wash membrane 2x5 minutes with PBS containing 0,5% Triton X-100
  2. Wash membrane 2x5 minutes with PBS
  3. Wash membrane 1x5 minutes with water


Mix the ECL mix, solution A and solution B 1:1 (ECL online manual pdf)

Add the ECL mix to the membrane and let it incubate for 1 minute, shake it so the whole membrane is constantly wet and then poor of and let the membrane dry
 
 

Exposing

  1. Mount membrane in exposure casettes.
  2. Add frame (top) to allow correct positioning of film relative to the rainbow marker for evaluation of labelling specificity.


In the dark room

  1. Place ECL Hyperfilm gently on the membrane. Notch at upper left. Film is kept in light secure container in fridge. Open container only in dark room.
  2. Close cassette. Locks should snap into place.
  3. Expose the film on several different exposure times (0-15min), to make sure getting the best result.
 Developing
  1. Place frame in developer (20°C): 4 minutes (5 min)
  2. Transfer to rinse (room temp: 24°C): 30 seconds
  3. Place in fixative  (24°C): 7 min (5-10 min)
  4. Transfer to rinse (room temp: 24°C): 10 min (5-10 min)

 
 
 
 
 
 
 
 
 

Blotting transfer buffer (BTB): 10X
100mM Tris Base 12,14g in 1l
1.0 M glycine 75,1g in 1l
H2O up to 1l

Transfer buffer:  3l 500 ml
10X BTB  300 ml 50 ml
Methanol (20% final) 600 ml 100 ml
H2O  2,1 l 350 ml