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Profiling inflammatory responses with microfluidic immunoblotting.

Chang HN, Leroueil PR, Selwa K, Gasper CJ, Tsuchida RE, Wang JJ, McHugh WM, Cornell TT, Baker JR, Goonewardena SN - PLoS ONE (2013)

Bottom Line: The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting.Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies.We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.

View Article: PubMed Central - PubMed

Affiliation: Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of these limitations, we have designed a microfluidic protein immunoblotting device to profile multiple signaling pathways simultaneously. We show the utility of this approach by profiling inflammatory signaling pathways (NFκB, JAK-STAT, and MAPK) in cell models and human samples. The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting. Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies. We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.

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Comparison of traditional and microfluidic immunoblotting in human blood monocyte samples.Representative immunoblots for RelA/p65 at four protein concentrations (5, 1, 0.5 and 0.1 µg) and three antibody dilutions (I: 1:1000; II: 1:1666; III: 1:5000) using (A) traditional and (B) microfluidic immunoblotting techniques. The signal intensity for the (C) traditional and (D) microfluidic blots were quantified using ImageJ software and normalized to the signal associated with 5 µg of protein probed with the 1:1000 p65 antibody dilution. Immunoblots are representative of three independent PVDF membranes from the same PBMC lysates.
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pone-0081889-g002: Comparison of traditional and microfluidic immunoblotting in human blood monocyte samples.Representative immunoblots for RelA/p65 at four protein concentrations (5, 1, 0.5 and 0.1 µg) and three antibody dilutions (I: 1:1000; II: 1:1666; III: 1:5000) using (A) traditional and (B) microfluidic immunoblotting techniques. The signal intensity for the (C) traditional and (D) microfluidic blots were quantified using ImageJ software and normalized to the signal associated with 5 µg of protein probed with the 1:1000 p65 antibody dilution. Immunoblots are representative of three independent PVDF membranes from the same PBMC lysates.

Mentions: We wished to compare the test characteristics of microfluidic immunoblotting with traditional immunoblotting. We collected peripheral blood monocytes (PBMC) from healthy volunteers and isolated protein lysates. Four different protein amounts were separated using gel electrophoresis and then transferred to a PVDF membrane. Using a primary antibody to RelA/p65, a member of the NF-κB transcriptional family, we compared the signals between microfluidic and traditional immunoblotting at three different primary antibody dilutions and the different protein loading amounts (Figure 2 and Figure S1). The microfluidic immunoblotting approach resulted in slightly lower, but comparable signal intensities compared to those from traditional immunoblotting. In both approaches, the signals and signal variation were comparable under the conditions tested.


Profiling inflammatory responses with microfluidic immunoblotting.

Chang HN, Leroueil PR, Selwa K, Gasper CJ, Tsuchida RE, Wang JJ, McHugh WM, Cornell TT, Baker JR, Goonewardena SN - PLoS ONE (2013)

Comparison of traditional and microfluidic immunoblotting in human blood monocyte samples.Representative immunoblots for RelA/p65 at four protein concentrations (5, 1, 0.5 and 0.1 µg) and three antibody dilutions (I: 1:1000; II: 1:1666; III: 1:5000) using (A) traditional and (B) microfluidic immunoblotting techniques. The signal intensity for the (C) traditional and (D) microfluidic blots were quantified using ImageJ software and normalized to the signal associated with 5 µg of protein probed with the 1:1000 p65 antibody dilution. Immunoblots are representative of three independent PVDF membranes from the same PBMC lysates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842271&req=5

pone-0081889-g002: Comparison of traditional and microfluidic immunoblotting in human blood monocyte samples.Representative immunoblots for RelA/p65 at four protein concentrations (5, 1, 0.5 and 0.1 µg) and three antibody dilutions (I: 1:1000; II: 1:1666; III: 1:5000) using (A) traditional and (B) microfluidic immunoblotting techniques. The signal intensity for the (C) traditional and (D) microfluidic blots were quantified using ImageJ software and normalized to the signal associated with 5 µg of protein probed with the 1:1000 p65 antibody dilution. Immunoblots are representative of three independent PVDF membranes from the same PBMC lysates.
Mentions: We wished to compare the test characteristics of microfluidic immunoblotting with traditional immunoblotting. We collected peripheral blood monocytes (PBMC) from healthy volunteers and isolated protein lysates. Four different protein amounts were separated using gel electrophoresis and then transferred to a PVDF membrane. Using a primary antibody to RelA/p65, a member of the NF-κB transcriptional family, we compared the signals between microfluidic and traditional immunoblotting at three different primary antibody dilutions and the different protein loading amounts (Figure 2 and Figure S1). The microfluidic immunoblotting approach resulted in slightly lower, but comparable signal intensities compared to those from traditional immunoblotting. In both approaches, the signals and signal variation were comparable under the conditions tested.

Bottom Line: The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting.Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies.We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.

View Article: PubMed Central - PubMed

Affiliation: Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of these limitations, we have designed a microfluidic protein immunoblotting device to profile multiple signaling pathways simultaneously. We show the utility of this approach by profiling inflammatory signaling pathways (NFκB, JAK-STAT, and MAPK) in cell models and human samples. The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting. Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies. We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.

Show MeSH