Immunoblotting and immunodetection.
This unit provides protocols for immunoblotting, which is used to identify specific protein sequences separated by electrophoresis and transferred to an appropriate membrane and recognized by a polyclonal or monoclonal antibody. After the proteins are separated on a gel, they are transferred to a membrane by electroblotting or a semidry transfer system. Proteins on the membrane can be visualized with the reversible stain Ponceau S to assess the completeness of transfer. Then the blot is analyzed with antibodies. The primary antibody is specific for the protein(s) of interest; the secondary antibody (an anti-Ig) is conjugated to horseradish peroxidase or alkaline phosphatase and detected colorimetrically or by chemiluminescence. The membrane can be stripped and reused for other probes.
Gallagher
S R
SR
Motorola Corporation, Tempe, Arizona, USA.
Winston
S E
SE
Fuller
S A
SA
Hurrell
J G
JG
D000818:Animals; D001391:Azo Compounds; D019921:Biotinylation; D015153:Blotting, Western / Q000295:instrumentation / Q000379:methods*; D004396:Coloring Agents; D006801:Humans; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D011506:Proteins / Q000032:analysis* / Q000276:immunology
D001391:Azo Compounds; D004396:Coloring Agents; D011506:Proteins; C032756:ponceau S