Irdye infrared dye conjugated secondary antibodies

Proteins were harvested by the use of RIPA Lysis Buffer and the protein concentration was determined by the use of the BCA Protein Assay Kit (both from Abcam, Cambridge, UK). After denaturation by boiling for 5 min, the samples were incubated on ice and then separated along with a commercial protein ladder by SDS-PAGE. A semi-dry system was used for the transfer of separated proteins in the gel to a PVDF membrane. The membrane was blocked by shaking in 3% BSA solution, followed by incubation with primary antibodies and thereafter with IRDye® infrared.
Dye-conjugated secondary antibodies (LI-COR Biosciences GmbH, Bad Homburg, Germany). The infrared intensity was measured by the use of an Odyssey CLx Infrared Imaging System (LI-COR Biosciences GmbH). The primary antibodies were rabbit polyclonal anti-LC3B and anti-P62 (Abcam, Cambridge, UK) and rabbit monoclonal anti-GAPDH antibody (Abcam).

After treatment, cells were lysed with RIPA lysis buffer (Abcam, Cambridge, UK), and total protein was purified by a standard protocol. Protein concentration was determined by the BCA Protein Assay Kit (Abcam, Cambridge, UK). Before SDS-PAGE separation, the samples were denatured by boiling for 5 min and then kept on ice. The separated proteins were transferred from the gel to a PVDF membrane by a semidry system. The membrane was blocked by incubation in 3% BSA solution, incubated with primary antibodies, washed, and incubated with IRDye® infrared dye-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany). The infrared intensity was measured with an Odyssey CLx Infrared Imaging System (LI-COR). Rabbit polyclonal antibodies against UHMK1 (PA550622, Invitrogen, Germany), GAPDH (Cell Signaling Technology, Danvers, MA, USA), and IRDye^® 800CW goat anti-rabbit IgG secondary antibody (LI-COR) were used.

The cells were lysed in RIPA lysis buffer (Abcam, Cambridge, UK), and total protein was isolated according to a standard protocol. The protein concentration was determined using the BCA Protein Assay Kit (Abcam, Cambridge, UK). Before SDS–PAGE separation, the samples were denatured by boiling for 5 min and then kept on ice. The separated proteins were transferred from the gel to a PVDF membrane using a semi-dry system. The membrane was blocked by incubation in 3% BSA solution and then incubated with primary antibodies, including a rabbit polyclonal antibody against LC3B (Cell Signaling Technology, Danvers, MA, USA, #2775), mouse monoclonal antibodies against SQSTM1/p62 (Cell Signaling Technology, Danvers, MA, USA, #5114), vimentin (ab V9, Abcam, Cambridge, UK, #ab8069), and β-actin (Sigma‒Aldrich, Munich, Germany, #A5441), and rabbit monoclonal antibodies against E-cadherin (ab 24E10, Cell Signaling Technology, Danvers, MA, USA, #3195) and GAPDH (Cell Signaling Technology, Danvers, MA, USA, #5174). After washing, the membranes were incubated with IRDye infrared dye-conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany). The infrared intensity was measured using an Odyssey CLx Infrared Imaging System (LI-COR).