Post transfer analysis
Post transfer analysis
After the transfer, proceed to immunodetection, store the membrane for future use, or
stain the membrane.
• For immunodetection of proteins, use the WesternBreeze
™
Chromogenic or
Chemiluminescent Immunodetection Kits available at thermofisher.com (see
“iBlot
™
2 Transfer Stacks“ on page 57–“Additional products“ on page 57), or
any other immunodetection kit.
Note:
When using the iBlot
™
2 Dry Blotting System to transfer proteins from
SDS-PAGE gels, the applied field strength can result in the partial depletion of
negative ions bound to the proteins. This may result in a slight decrease in the
amount of protein migrating from the gel, but it also results in improved binding
of the transferred proteins to the membrane. Since the membrane maintains the
protein load better, higher sensitivity can be achieved for subsequent
immunodetection procedures.
• To store nitrocellulose membranes, air-dry the membrane and store the
membrane in an air-tight plastic bag at room temperature or 4
℃
. Avoid storing
nitrocellulose at temperatures below –20
℃
. Low temperatures cause the
nitrocellulose to turn brittle.
• To store PVDF membranes, air-dry the membrane and store the membrane in an
air-tight plastic bag at room temperature, 4
℃
, or –80
℃
. When you are ready to
use the membrane, re-wet the membrane with methanol for a few seconds, then
rinse the membrane thoroughly with deionized water to remove methanol.
• To stain membranes after blotting, use any method of staining for total protein
visualization, such as Coomassie
™
Blue R-250, Ponceau S, Amido Black, Novex
™
Reversible Membrane Protein Stain Kit, or SYPRO
™
Ruby
™
Blot Stain
(“Additional products“ on page 57). The iBlot
™
2 Gel Transfer Device blotting
protocol is compatible with most of the staining methods listed above.
Note:
The sensitivity of total protein membrane staining after blotting with the
iBlot
™
2 Gel Transfer Device is slightly lower than the total membrane protein
staining obtained with the semi-wet transfer protocol. However, due to the nature
of dry blotting, lower transfer does not affect the immunodetection sensitivity.
If you do not detect any proteins on the membrane after immunodetection or staining,
refer to Troubleshooting “Introduction“ on page 45. Refer to the manufacturer
recommendations for optimizing immunodetection.
The immunodetection profile of proteins transferred using the iBlot
™
2 Dry Blotting
System may differ from what is observed when using other transfer methods, such as
traditional semi-dry or wet blotting systems. It is recommended to optimize
parameters such as gel protein load, primary and secondary antibody dilution, and
exposure time (see “Optimizing blotting“ on page 49 for details) when using the
iBlot
™
2 Dry Blotting System for the first time with any new combination of antigen
and detection reagents.
B
48
iBlot
™
2 Dry Blotting System User Guide
