Biology Reference
In-Depth Information
9. When the gel is taken off from the glass plates, take care not to
break the gel. Cut the acidic corner of the gel to see its
direction.
10. Air bubbles between the gel and membrane should be removed.
Change the time and/or the electric current, depending on
the target range of molecular weight. Semi-dried transfer sys-
tems may also be used for blotting proteins to the membrane,
while we have obtained good results using tank transfer sys-
tems. After the electrical transfer, cut the acidic corner of the
membrane to see its direction.
11. Incubate the membrane face down so as to prevent drying of
the surface of the membrane. After the incubation, check
whether the entire surface of the membrane is stained blue.
12. The membrane should be incubated with more than 20 mL of
serum, suffi cient to cover the entire surface of the membrane.
Take care to avoid drying of the surface of the membrane. The
serum dilution could be changed depending on the total
amount of IgE. Usually, we use 100-fold diluted serum
(Fig.
2
).
13. After incubation, the serum solution must be sterilized with
1 % sodium hypochlorite before it is disposed of.
14. ECL plus (plex) reagent should cover the entire surface of the
membrane. Use of excessive reagent or a long reaction time
might result in excessive chemical reaction, resulting in void
spots. Dilution of the reagent with ultrapure water or a short
reaction time might yield better results.
15. Insuffi cient water may cause drying of the surface of the mem-
brane. Slowly put the glass plate down on the water hill to
allow the water to spread over the entire surface of the mem-
brane. Spotty air bubbles between the membrane and the glass
plate would yield unclear images.
Acknowledgments
This work was supported by a grant from the Ministry of Health,
Labour and Welfare, Japan.
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