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    Optimization of western blotting for the detection of proteins of different molecular weight

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    Author
    Heda, G.D.
    Shrestha, L.
    Thapa, S.
    Date
    2020
    Journal
    BioTechniques
    Publisher
    Eaton Publishing Co.
    Type
    Article
    
    Metadata
    Show full item record
    See at
    https://doi.org/10.2144/btn-2019-0124
    Abstract
    Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0-20%) in Towbin's transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. However, for medium-sized (LAMP1) and small (Rab11a) proteins, a lower concentration of methanol (10%) was sufficient to produce a maximal signal. Therefore, methanol, a toxic solvent, can be removed from or reduced in TTB without compromising signal strength. Here, we show modifications that may be useful in detecting and/or improving the signal of low-abundance proteins.
    Keyword
    blocking agents
    CFTR
    LAMP1
    methanol concentration
    optimization
    Rab11a
    Towbin's transfer buffer
    western blotting
    Identifier to cite or link to this item
    https://www.scopus.com/inward/record.uri?eid=2-s2.0-85086524487&doi=10.2144%2fbtn-2019-0124&partnerID=40&md5=8663cf107a075152dcfbcc017243a310; http://hdl.handle.net/10713/13133
    ae974a485f413a2113503eed53cd6c53
    10.2144/btn-2019-0124
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