scholarly journals Single-cell mobility shift electrophoresis reports protein localization to the cell membrane

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 972-979 ◽  
Author(s):  
Elly Sinkala ◽  
Elisabet Rosàs-Canyelles ◽  
Amy E. Herr
Keyword(s):  
Single Cell ◽  
Gel Electrophoresis ◽  
Protein Localization ◽  
Single Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mobility Shift ◽  
Receptor Proteins ◽  
Cell Mobility ◽  
Regulatory Systems ◽  
Surface Receptor

Prepending surface receptor immunostaining with single-cell polyacrylamide gel electrophoresis provides a new tool with which to understand how localization of surface receptor proteins controls the complex regulatory systems in single cells.

SIFTER: Electrophoretic complex fractionation protocol

2021 ◽  
Author(s):  
Julea Vlassakis ◽  
Louise L Hansen ◽  
Amy E Herr
Keyword(s):  
Single Cell ◽  
Protein Interaction ◽  
Gel Electrophoresis ◽  
Protein Complexes ◽  
Single Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Simultaneous Detection ◽  
Single Cell Protein ◽  
Cell Protein ◽  
A Cell

Abstract We introduce micro-arrayed, differential detergent fractionation for the simultaneous detection of protein complexes in 100s of individual cells with SIFTER (Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout). Size-based fractionation of protein complexes is accomplished with five assay steps. First, a cell suspension generated by trypsinization is introduced onto a microwell array, and single cells are settled into the microwells by gravity. Cells are lysed in F-actin stabilization buffer that is delivered by a hydrogel lid. Second, the protein complexes are fractionated from the smaller monomers by polyacrylamide gel electrophoresis. Monomers are electrophoresed into the gel and are immobilized using a UV-induced covalent reaction to benzophenone. Third, a protein-complex depolymerization buffer is introduced by another hydrogel lid. Fourth, the recently depolymerized complexes are electrophoresed into a region of the gel separate from the immobilized monomers, where the complex fraction are in turn immobilized. Fifth, in-gel immunoprobing detects the immobilized populations of monomer and depolymerized complexes. These general steps are built on previously published protocols for bulk actin studies, single-cell western blotting, and bidirectional separations1-4.

scholarly journals μCB-seq: Microfluidic cell barcoding and sequencing for high-resolution imaging and sequencing of single cells

2020 ◽  
Author(s):  
Tyler N. Chen ◽  
Anushka Gupta ◽  
Mansi Zalavadia ◽  
Aaron M. Streets
Keyword(s):  
High Resolution ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Protein Localization ◽  
Single Cells ◽  
Optical Measurements ◽  
Detection Sensitivity ◽  
High Resolution Imaging ◽  
Cell Analysis ◽  
Resolution Imaging

AbstractSingle-cell RNA sequencing (scRNA-seq) enables the investigation of complex biological processes in multicellular organisms with high resolution. However, many phenotypic features that are critical to understanding the functional role of cells in a heterogeneous tissue or organ are not directly encoded in the genome and therefore cannot be profiled with scRNA-seq. Quantitative optical microscopy has long been a powerful approach for characterizing diverse cellular phenotypes including cell morphology, protein localization, and chemical composition. Combining scRNA-seq with optical imaging has the potential to provide comprehensive single-cell analysis, allowing for functional integration of gene expression profiling and cell-state characterization. However, it is difficult to track single cells through both measurements; therefore, coupling current scRNA-seq protocols with optical measurements remains a challenge. Here, we report Microfluidic Cell Barcoding and Sequencing (μCB-seq), a microfluidic platform that combines high-resolution imaging and sequencing of single cells. μCB-seq is enabled by a novel fabrication method that preloads primers with known barcode sequences inside addressable reaction chambers of a microfluidic device. In addition to enabling multi-modal single-cell analysis, μCB-seq improves gene detection sensitivity, providing a scalable and accurate method for information-rich characterization of single cells.

scholarly journals Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex.

1990 ◽  
Vol 10 (3) ◽  
pp. 1249-1253 ◽  
Author(s):  
S Fujiwara ◽  
S Koizumi ◽  
R J Fisher ◽  
N K Bhat ◽  
T S Papas
Keyword(s):  
T Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Mobility Shift ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex

1990 ◽  
Vol 10 (3) ◽  
pp. 1249-1253
Author(s):  
S Fujiwara ◽  
S Koizumi ◽  
R J Fisher ◽  
N K Bhat ◽  
T S Papas
Keyword(s):  
T Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Mobility Shift ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

scholarly journals Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages.

1995 ◽  
Vol 15 (7) ◽  
pp. 3579-3586 ◽  
Author(s):  
A Eilers ◽  
D Georgellis ◽  
B Klose ◽  
C Schindler ◽  
A Ziemiecki ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Phosphorylation ◽  
Gamma Activation ◽  
Mobility Shift ◽  
Two Dimensional Gel Electrophoresis ◽  
Ifn Gamma ◽  
Activation Site

Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-gamma activation factor (GAF). GAF is formed after phosphorylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell line U937 into monocytes increases the amount of cellular GAF after IFN-gamma treatment and at the same time increases the phosphorylation of STAT1. Here we show that activation of the JAK family kinases, which are instrumental in mediating STAT1 phosphorylation on tyrosine, did not increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually identical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosphoamino acids and mapping of phosphopeptides showed an IFN-gamma-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiation-induced shift toward more acidic isoforms. All isoforms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift typical for tyrosine-phosphorylated STAT1. Consistent with the importance of Ser phosphorylation for high-affinity binding to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF complexes in an electrophoretic mobility shift assay. Our data indicate that the activity of GAF is modulated by STAT1 serine kinases/phosphatases and suggest that this mechanism is employed in the developmental control of macrophage responsiveness to IFN-gamma.

Protein Mapping and Characterization of Post-Translational Modifications of Ku86 Protein Variants in Multiple Myeloma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3510-3510
Author(s):  
Geraline Cow ◽  
Charles Gullo ◽  
Feng Ge ◽  
Gerrard Teoh
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Gel Electrophoresis ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Proteolytic Cleavage ◽  
Regulatory Subunit ◽  
Mobility Shift ◽  
N Terminus

Abstract A single biological event, immunoglobulin heavy chain (IgH) isotype class switch recombination (CSR) is thought to mark the onset of multiple myeloma (MM). The DNA repair enzyme, DNA-dependent protein kinase (DNA-PK), which consists of a heterodimeric regulatory subunit (Ku70/Ku86) and its catalytic subunit (DNA-PKcs), is the principal mediator of IgH isotype CSR. Studies in Ku70 and Ku86 knockout mice have suggested that Ku proteins could have a role to play in carcinogenesis. Since we have previously detected truncated variants of Ku86 (Ku86v) in 86% to 100% of freshly isolated patient MM cells, we therefore hypothesize that Ku86v could be a putative oncoprotein. Two variants of Ku86 are predominantly found in MM cells: a 69 kDa variant truncated at the C-terminus (Ku86v-N), which has lost its DNA repair function; and a 56 kDa N-terminus truncated variant (Ku86v-C), which is not translocated into the nucleus and aberrantly expressed on the cell membrane. Our prior studies using Northern blotting, whole cell polyacrylamide gel electrophoresis (PAGE) and electrophoretic mobility shift assays (EMSA) have demonstrated that Ku86vs in MM cells are not the result of alternative RNA splicing; or in vitro degradation, as has been described in human lymphocytes. Moreover, since many proteins in MM cells are known to be heavily glycosylated, and deglycosylation could lead to shorter forms of the protein, we now show using Endo H digestion that Ku86vs are also not the result of N-linked deglycosylation. Rather, Ku86vs are formed by in vivo proteolytic cleavage via a trypsin-like serine protease. Since the proteasome degradation pathway is highly active in MM cells; and protease digestion of DNA-PK holoenzyme is enhanced by proteasome inhibition (i.e. bortezomib treatment), these data support the notion that in vivo activation of putative Ku86v oncoproteins within MM cells could arise via a constitutively active post-translational proteolytic process. To define a functional role for Ku86v, we first demonstrate that Ku86v is physically associated with the most abundant anti-apoptotic protein in MM cells, BCL2. We then used cyanogen bromide immunoprecipitation to purify BCL2-bound Ku86vs followed by 2-dimensional gel electrophoresis (2DGE) to resolve these purified proteins, and demonstrate that only the 56 kDa Ku86v-C (pI 5.0) bound to BCL2. Both full length Ku86 and Ku86v-N were not detected. We then isolated Ku86v-C from the 2D gel and preliminary mass spectrometric analyses suggest that the N-terminus truncation retains the Ku autoantigen related protein 1 (KARP1) motif of Ku86. The significance of this is still under investigation. In conclusion, we have found that Ku86v-C (56 kDa pI 5.0) is formed by post-translational proteolytic cleavage. Since, Ku86v-C is significantly associated with BCL2 oncoprotein, we therefore speculate that Ku86v-C could possibly potentiate BCL2’s anti-apoptotic function. Accordingly, Ku86v-C could be considered as a potential target for therapeutic intervention in MM.

A COMPARATIVE STUDY OF SEED PROTEINS OF ALLOPOLYPLOIDS OF GOSSYPIUM BY GEL ELECTROPHORESIS

1971 ◽  
Vol 13 (1) ◽  
pp. 155-158 ◽  
Author(s):  
J. P. Cherry ◽  
F. R. H. Katterman ◽  
J. E. Endrizzi
Keyword(s):  
Comparative Study ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gene Mutations ◽  
Seed Proteins ◽  
Polyacrylamide Gel ◽  
Synthetic Mixture ◽  
Banding Patterns ◽  
Regulatory Systems ◽  
Species Specific

Polyacrylamide gel electrophoresis of seed proteins from recently synthesized F1 triploid and colchicine-induced hexaploid plants showed the additive banding patterns of their parents (G. hirsutum × G. sturtianum) when compared to the synthetic mixture of the latter. Gene mutations, diploidization and species specific regulatory systems are discussed as possible evolutionary processes for the protein banding differences in the allopolyploids of the genus Gossypium.

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