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2016 ; 11
(8
): 1508-30
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Single cell-resolution western blotting
#MMPMID27466711
Kang CC
; Yamauchi KA
; Vlassakis J
; Sinkala E
; Duncombe TA
; Herr AE
Nat Protoc
2016[Aug]; 11
(8
): 1508-30
PMID27466711
show ga
This protocol describes how to perform western blotting on individual cells to
measure cell-to-cell variation in protein expression levels and protein state.
Like conventional western blotting, single-cell western blotting (scWB) is
particularly useful for protein targets that lack selective antibodies (e.g.,
isoforms) and in cases in which background signal from intact cells is
confounding. scWB is performed on a microdevice that comprises an array of
microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer
functions as both a molecular sieving matrix during PAGE and a blotting scaffold
during immunoprobing. scWB involves five main stages: (i) gravity settling of
cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE
of each single-cell lysate; (iv) exposure of the gel to UV light to blot
(immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of
immobilized proteins. Multiplexing can be achieved by probing with antibody
cocktails and using antibody stripping/reprobing techniques, enabling detection
of 10+ proteins in each cell. We also describe microdevice fabrication for both
uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of
small pore size, we describe an optional gel de-cross-linking protocol for more
effective introduction of antibodies into the gel layer. Once the microdevice has
been fabricated, the assay can be completed in 4-6 h by microfluidic novices and
it generates high-selectivity, multiplexed data from single cells. The technique
is relevant when direct measurement of proteins in single cells is needed, with
applications spanning the fundamental biosciences to applied biomedicine.
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free
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