The development of microfluidic-based western blotting: Technical advances and future perspectives

Desire, Christopher T.; Arrua, R. Dario; Strudwick, Xanthe L.; Kopecki, Zlatko; Cowin, Allison J.; Hilder, Emily F.

doi:10.1016/j.chroma.2023.463813

Cited by 4 publications

(2 citation statements)

References 154 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this way, it provides information on the purity of the sample. This contrasts with other techniques, where non-specific binding can significantly compromise the validity of the results [ 7 , 8 ]. However, Western blotting (especially the manual ones) may carry several errors from sample preparation to signal processing.…”

Section: Introductionmentioning

confidence: 99%

“…One of the major limiting factors in the case of Western blot is the transfer or blotting step [ 10 ], which is also known to be highly variable depending on the membrane type and porosity that influences the transfer efficiency of proteins with different molecular weights (between 60% and 100%) for some hydrophobic membranes, thus making quantification difficult. This is a particularly problematic step for large molecular weight proteins that can be difficult to transfer effectively [ 7 , 11 ]. In addition, quite often a large amount of sample (5–15 µg) would be needed for adequate results [ 12 ], which can jeopardize the analysis when the amount of sample is limited [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Multiplexed Fluorescence Plate Reader In Situ Protein Expression Assay in Apoptotic HepG2 Cells

Jakabfi-Csepregi

Kovaćs

Kaltenecker

et al. 2023

IJMS

View full text Add to dashboard Cite

Instead of Western blot being considered as a gold standard for intracellular protein expression assays, we developed a novel multiplexed high throughput (180 tests/day) in situ manual protein expression method directly in 96-well plates using 25,000–100,000 cells/well after formaldehyde fixation and Triton X 100 permeabilization. HepG2 cells were treated with ochratoxin A (OTA) and staurosporine (STP) to induce apoptosis. Antioxidant and apoptotic cell signaling protein expression were studied by various rabbit primary antibodies and HRP labeled secondary antibodies. The HRP labeled immune complexes were developed by H2O2/Ampliflu Red fluorogenic reagent and measured in a plate reader. Our assay can simultaneously quantify 22 protein antigens in one plate with 4 technical replicates with an interassay imprecision of <10% CV. The fluorescence signals are referred to total intracellular protein contents in the wells and given as fluorescence/protein ratio FPR, expressed as % of the controls (FPR %). OTA caused a dose–response increase (p < 0.05–p < 0.001) in SOD2, CAT, ALB, CASP3,7,9, BCL2, BAX, Nf-kB, phospho-Erk1/2/Erk1/2, phospho-Akt/Akt, phospho-p38/p38, and phospho-PPARg/PPARg levels while phospho-AMPK/AMPK ratios decreased (p < 0.05–p < 0.001). On the contrary, STP induced a dose–response decrease (p < 0.05–p < 0.001) in CASP3,7,9, BAX, BCL2, Nf-kB and phospho-Erk1/2/Erk1/2 expression while B-ACT, phospho-Akt/Akt, phospho-p38/p38 and phospho-PPARg/PPARg ratios increased.

show abstract

Section: Introductionmentioning

confidence: 99%