25
sandwich
Western detection system not working or not sensitive enough
•
Include proper positive or negative control antigen.
Consult kit manual.
•
Use protein markers with coloured reference bands during
PAGE.
•
Stain gel with Coomassie, or stain membrane with
Ponceau S.
Transfer time too short
–
increase transfer time
Power setting too low
•
Increase Voltage
•
Buffer may be prepared improperly
–
prepare new buffer
and increase voltage.
Charge-to-mass ratio incorrect for native transfers.
•
Proteins close to isoelectric point (pI). Change buffer pH so
that it is at least 2 pH unit higher or lower than pI of protein
of interest.
Defective or inappropriate power supply used.
•
Check fuse of power supply. Ensure max. current output of
power supply is at least 2000mA.
Excessive methanol restricting transfer.
•
Reduce methanol concentration to maximize protein
transfer from gel, but without reducing concentration to
the extent that it prevents binding to nitrocellulose.
Alternatively reduce methanol concentration and switch
to PVDF.
Protein precipitating in gel
•
Use SDS in transfer buffer (SDS can increase transfer
efficiency, but it can also reduce nitrocellulose binding
affinity and affect protein-antibody reactivity).
•
Remove alcohol from transfer buffer.
Swirls or missing bands;
diffuse transfers
Poor gel-membrane contact. Air bubbles or excess buffer
remain between membrane and gel.
•
Carefully remove air bubbles between gel and membrane
using a rolling pin
•
Use more, or thicker, filter paper in gel-membrane
sandwich
•
Replace the fibre pads, as they degrade and remain
permanently compressed over time.
Problem with gel electrophoresis.
•
Poor gel polymerization, inappropriate running conditions,
buffer contamination, excessive sample application all
contribute to poor quality gels and transfers.
Gel cassette pattern
transferred to blot
Contaminated fibre pads
•
Replace fibre pads or clean thoroughly.
Contaminated transfer buffer
•
Replace buffer solutions.
Poor binding to membrane
Excessive methanol restricting transfer.