Irdye 680lt anti mouse igg antibody

Proteins were quantitated (Epoch Gen5, BioTek, Winooski, VT, USA), and mixed at
1:1 with SDS sample buffer. Ten micrograms of protein were loaded on a
SDS-polyacrylamide gel electrophoresis of various concentrations depending on size
of target protein, and ran at a constant current. After transfer, the membrane
(Immobilon-P PVDF, Millipore, Billerica, MA, USA) was used for immunostaining.
Anti-α-actin mouse monoclonal antibody (AC-15, Sigma) was simultaneously
added with the antibody to the target protein. The membrane was further incubated
with IRDye 800CW-labeled secondary antibody for target protein and IRDye 680LT
anti-mouse IgG antibody (LI-COR, Lincoln, NE, USA). The membrane was
simultaneously scanned at both wave lengths on an infrared fluorescence scanner
(Odyssey, LI-COR), with target protein as green and control α-actin as
red.

Various proteins, include crude homogenate of cortex and isolated glomeruli were quantitated (Epoch Gen5, BioTek, Winooski, VT), and mixed at 1:1 with SDS sample buffer following our published method [20 (link)]. The membrane was incubated with IRDye®800CW labeled secondary antibody for target protein and IRDye® 680LT anti-mouse IgG antibody (LI-COR, Lincoln, NE). The membrane was simultaneously scanned at both wave lengths on an infrared fluorescence scanner (Odyssey, LI-COR), with target protein as green and control α-actin as red. Each band was digitally quantitated as IOD.