Cell lysates were prepared and protein concentration determined as described[5 (link)]. Samples containing equivalent amounts of cellular proteins (30-50 μg/sample) were fractionated in 10% pre-cast gels (Perbio Science, Mordialloc, Australia) then transferred to hybond C extra nitrocellulose membranes (Millipore, Bedford, MA). Membranes were blocked with 2.5% bovine serum albumin (BSA) in Tris buffered saline containing 0.1% Tween (TBST). Immunoblotting was performed as described[5 (link)] using primary antibodies directed against ERK1/2, phospho-ERK1/2, p38, phospho-p38, phospho-serine 536 NF-κB p65, and ß-actin (Cell Signalling, Danvers, MA). Densitometry ratios were normalized to appropriate loading control content and results expressed as relative density.
Hybond c extra nitrocellulose membranes
Manufactured by Merck Group
Sourced in Australia
Hybond C extra nitrocellulose membranes are a type of laboratory equipment used in various biomedical and biochemical applications. These membranes are designed for the transfer and immobilization of proteins, nucleic acids, and other macromolecules during analytical techniques such as Western blotting, Northern blotting, and Southern blotting.
Automatically generated - may contain errors
2 protocols using hybond c extra nitrocellulose membranes
1
Western Blot Analysis of Signaling Proteins
2
PD-L1 Expression in NSCLC Cell Lines
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For Western blot analysis, proteins were extracted from the following NSCLC cell lines: H549, H23, H1299, and A549. Aliquots of 30 mL were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (4%-12%) and transferred to Hybond-Cextra nitrocellulose membranes (Milipore, Bedford, MA). The membranes were blocked in phosphate-buffered saline-Tween-20 (0.03%) with 5% milk at room temperature (for 1 hour) and incubated with anti-PD-L1 antibody (Cell Signaling Technology, Danvers, MA) and anti-glyceraldehyde-3phosphate dehydrogenase (Santa Cruz) at room temperature for 1 hour, followed by incubation in horseradish peroxidase-conjugated secondary antibodies.
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