scholarly journals Mesowestern Blot: Simultaneous Analysis of Hundreds of Sub-Microliter Lysates

2021 ◽  
Author(s):  
Cameron O Zadeh ◽  
Jonah R Huggins ◽  
Baylee C Westbury ◽  
William R Interiano ◽  
S Ashley Phillips ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Western Blotting ◽  
Limit Of Detection ◽  
Scale Up ◽  
Barriers To Adoption ◽  
Western Blots ◽  
Post Translational Modifications ◽  
Typical Cell ◽  
Weight Protein ◽  
Biology Laboratory

Western blotting is a widely-used technique for molecular-weight-resolved analysis of proteins and their post-translational modifications, but has been refractory to affordable scale-up. Here, we report the Mesowestern blot, which uses a 3D-printable gel-casting mold to enable affordable, high-throughput Western blotting with standard sample preparation and small (<1 uL) sample sizes. The casted polyacrylamide gel contains 336, 0.5 uL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution range. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder as well as cell lysate (RIPA buffer) demonstrate protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semi-dry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is approximately 2 orders of magnitude, with a limit of detection (for beta-actin) of around 30 ng of total protein from mammalian cell lysates (~30-3000 cells). Because the gel mold is 3D-printable, users have significant design freedom for custom layouts, and there are few barriers to adoption by the typical cell and molecular biology laboratory already performing Western blots.

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

BioTechniques ◽  
2020 ◽  
Vol 68 (6) ◽  
pp. 318-324
Author(s):  
Ghanshyam D Heda ◽  
Lisa Shrestha ◽  
Sagarina Thapa ◽  
Shreya Ghimire ◽  
Diptika Raut
Keyword(s):  
Molecular Weight ◽  
Western Blotting ◽  
Low Molecular Weight ◽  
Transmembrane Conductance Regulator ◽  
High Molecular Weight Protein ◽  
Transmembrane Conductance ◽  
Weight Protein ◽  
Optimized Conditions ◽  
Cystic Fibrosis Transmembrane ◽  
Low Molecular Weight Proteins

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.

scholarly journals The protein antigens secreted in vivo by adult male Schistosoma mansoni

Parasitology ◽  
1997 ◽  
Vol 114 (3) ◽  
pp. 245-255 ◽  
Author(s):  
L. CUTTS ◽  
R. A. WILSON
Keyword(s):  
Molecular Weight ◽  
Western Blotting ◽  
Cellular Level ◽  
Protein Antigens ◽  
Parasite Protein ◽  
Western Blots ◽  
Portal Veins ◽  
Secretory Products ◽  
Abundance And Distribution

Adult schistosomes can be transferred surgically from donor C57BL/6 mice to the portal veins of naive recipients with complete success. This procedure bypasses larval development and the antibody response of the host is directed against, and can be used to identify, those antigens released only by viable, mature parasites. Serum collected from the recipient mice (WTS) was used in Western blotting studies to probe fractionated parasite protein. Twelve immunodominant proteins were identified, ranging in molecular weight from 14 to 208 kDa. The magnitude of the IgG response against each antigen could be divided into 2 categories, on the basis of optical densitometry of the blots. In addition, defined parasite fractions were probed with WTS by Western blotting, in order to determine the relative abundance and distribution of each antigen in schistosome tissue. To confirm and expand on these initial observations, oligospecific polyclonal antibody for each immunogen was affinity purified from Western blots; it was then used in immunocytochemistry to identify the sources of secretion for 8 of the 12 antigens, at the cellular level. From the results, it appeared that after the transfer of adult worms, the first antibodies detected were mostly directed against the gastrodermis. At later times additional reactivity was expressed against the tegumental membrane. These differences probably reflect the relative abundances of the gut and tegumental secretory products.

Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

1993 ◽  
Vol 70 (03) ◽  
pp. 438-442 ◽  
Author(s):  
B Grøn ◽  
C Filion-Myklebust ◽  
S Bjørnsen ◽  
P Haidaris ◽  
F Brosstad
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Isoelectric Point ◽  
Factor Xiii ◽  
Cross Linking ◽  
2D Electrophoresis ◽  
Complex Phenomenon ◽  
Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

1742-P: Targeting the Low Molecular Weight Protein Tyrosine Phosphatase for Obesity-Associated Diabetes Therapy

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 1742-P
Author(s):  
STEPHANIE M. STANFORD ◽  
MICHAEL A. DIAZ ◽  
JIWEN J. ZOU ◽  
ROBERT J. ARDECKY ◽  
ANTHONY PINKERTON ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Low Molecular Weight ◽  
Molecular Weight Protein ◽  
Diabetes Therapy ◽  
Protein Tyrosine ◽  
Weight Protein ◽  
Low Molecular Weight Protein

scholarly journals Comparison of the sensitivity of Western blotting between PVDF and NC membranes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yufang Xiang ◽  
Yuanyuan Zheng ◽  
Shaobo Liu ◽  
Gang Liu ◽  
Zhi Li ◽  
...  
Keyword(s):  
Western Blotting ◽  
Polyvinylidene Fluoride ◽  
Staining Intensity ◽  
Detection Sensitivity ◽  
Key Factors ◽  
Post Translational Modifications ◽  
Factors Affecting ◽  
Molecular Weights ◽  
Direct Blue ◽  
The Relationship

AbstractWestern blotting (WB) is one of the most widely used techniques to identify proteins as well as post translational modifications of proteins. The selection of electroblotted membrane is one of the key factors affecting the detection sensitivity of the protein which is transferred from gel to membrane in WB. The most common used membranes are polyvinylidene fluoride (PVDF) and nitrocellulose (NC) membranes. Which membrane of these two is more suitable for WB has not been reported so far. Here, by incubating proteins which were transferred to PVDF or NC membranes with a series of antibodies and different types of lectins, we investigated the relationship between the binding ability of these two membranes to proteins or glycoproteins and the molecular weight of the target protein. The antibody re-probed ability of the two membranes was also explored. Moreover, we verified the above results by directly incubating proteins having different molecular weights onto PVDF or NC membranes. Bound proteins were stained with direct blue-71, and the staining intensity was quantitated by scanning and densitometry.

Detection of inducible nitric oxide synthase inSchistosoma japonicumandS. mansoni

2004 ◽  
Vol 78 (1) ◽  
pp. 47-50 ◽  
Author(s):  
X.-C. Long ◽  
M. Bahgat ◽  
K. Chlichlia ◽  
A. Ruppel ◽  
Y.-L. Li
Keyword(s):  
Nitric Oxide ◽  
Molecular Weight ◽  
Inducible Nitric Oxide Synthase ◽  
Mouse Liver ◽  
Apparent Molecular Weight ◽  
Western Blots ◽  
Oxide Synthase ◽  
Host Tissues ◽  
Inducible Nitric Oxide

AbstractSchistosoma japonicumandS. mansoniwere tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental inS. japonicumand parenchymal inS. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme ofS. japonicumhad an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite–host interrelation remains to be clarified.

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