scholarly journals An Inexpensive Staining Alternative for Gelatin Zymography Gels

2019 ◽  
Vol 2 (3) ◽  
pp. 61 ◽  
Author(s):  
Wechselberger ◽  
Doppler ◽  
Bernhard
Keyword(s):  
Sodium Dodecyl ◽  
Gelatin Zymography ◽  
Staining Procedure ◽  
Electrophoretic Method ◽  
Resource Limited ◽  
Coomassie Blue ◽  
Staining Protocol ◽  
Proteolytic Activities ◽  
Ponceau S ◽  
Blue Background

Zymography is a widely used electrophoretic method to determine proteolytic activities in samples from various sources. The method is based on copolymerizing a suitable protein substrate within a sodium dodecyl sulfate-polyacrylamide gel. Following electrophoretic separation of the protease containing samples and a suitable incubation period, degradation of the substrate can be visualized through staining with Coomassie blue. Sites of proteolysis become visible as white bands on a dark blue background. However, this staining protocol requires considerable amounts of ethanol and acetic acid to remove unbound dye molecules. In this report, we describe a new staining protocol using Ponceau S which offers substantial advantages in terms of assay usability and cost reduction, especially when performing large quantities of zymograms or in resource-limited settings. Fast and reproducible staining of zymograms with our protocol is demonstrated, and reliable quantitation of proteolytic activity in comparison to the standard Coomassie staining procedure is shown.

Equine synovial fluid protein equalization via combinatorial peptide ligand libraries

2021 ◽  
Vol 1 (4) ◽  
pp. 18-22
Author(s):  
Pablo Fueyo ◽  
Marco Galleguillos ◽  
Cristóbal Dörner ◽  
Pedro A. Smith ◽  
Francisca Godoy ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Sodium Dodecyl ◽  
Electrophoretic Pattern ◽  
Peptide Ligand ◽  
Joint Pathology ◽  
Coomassie Blue ◽  
Standard Procedures ◽  
Sds Page ◽  
New Biomarkers ◽  
Synovial Fluid Protein

To gain further knowledge of the equine synovial fluid (SF) proteome, we propose a protocol based on the equalization of the relative concentrations of its proteins, which leads to the modification of the standard electrophoretic pattern revealing low-abundance proteins that otherwise remain undetected. Fresh SF samples were collected from ten macroscopically normal metacarpophalangeal joints of crossbred horses. The samples were processed using standard procedures as the control and via combinatorial peptide ligand libraries (CPLL) using low ionic forces (NaH2PO4 10 mM) at different pHs (4.0, 7.0, and 9.3) with 10% sodium dodecyl sulfate (SDS) and 25 mM DTT for protein resolubilization. Proteins were then separated by conventional 8% SDS-PAGE and stained with coomassie blue. After separation of the equalized proteins, there was a significant reduction in the albumin (the most abundant protein in the SF) and, at the same time, other protein bands arise that were not visible without CPLL processing. In addition, there was variation in the protein profiles at different pHs. The results suggest that protein equalization of the equine SF by CPLL could be a useful tool to better understand the articular homeostasis and/or for the detection of new biomarkers of joint pathology.

scholarly journals Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
N Kieffer ◽  
B Boizard ◽  
D Didry ◽  
JL Wautier ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Blue Staining ◽  
Igg Antibody ◽  
Immune Disorder ◽  
Immunochemical Characterization ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

Effect Of Storage On Surface Glycoproteins And Cyto-Skeletons Of Platelets

1981 ◽  
Author(s):  
A K Rao ◽  
G P Tuszynski ◽  
L Knight ◽  
J Willis ◽  
C Beckett
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Blue Staining ◽  
In Vitro Tests ◽  
Membrane Glycoproteins ◽  
Autologous Plasma ◽  
Coomassie Blue ◽  
Functional Defect

Platelets stored as concentrates (PC) at 22° C for 72 hours develop a functional defect in vitro tests. Alterations in membrane glycoproteins of platelets have been shown to effect platelet function. We have investigated the effect of storage on membrane glycoproteins (GP) and cytoskeletons (cyto.) of platelets. Gel filtered platelets from fresh PC were labeled with 125Iodine by Iodogen technique and gel filtered again to remove free iodide. Platelets were concentrated by albumin density gradient centrifugation, resuspended in autologous plasma and stored for 72 hours at 22° C. Aliquots of fresh and stored PC were solubilized with 2% sodium dodecyl sulphate (SDS) containing 5% mercaptoethanol and subjected to polyacrylamide gel electrophoresis (PAGE). In one experiment, separate aliquots of fresh and stored platelets were labeled and similarly analyzed. Gels were stained with Coomassie blue and subjected to autoradiography. Coomassie blue staining did not reveal major differences between fresh and stored platelets. Autoradiography revealed a decrease in the 170,000 dalton surface protein (GP-I) of platelets after storage. Triton insoluble cyto. of thrombin activated fresh and stored platelets were solubilized with SDS and analyzed by PAGE and autoradiography. Cytoskeletons from fresh PC revealed the presence of a 110,000 dalton surface protein (GP-III). However, cyto. from similarly treated stored platelets showed a markedly decreased amount of this protein. Thus stored platelets have decreased amounts of the 170,000 dalton surface protein (GP-I) along with decreased amounts of the 110,000 dalton protein (GP-III) associated with the cyto. of thrombin activated platelets. These changes may contribute to the functional defect reported in stored platelets.

scholarly journals Platelet antithrombin defect in malignancy: platelet protein alterations

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 479-485 ◽  
Author(s):  
EM Sloand ◽  
DM Kenney ◽  
FC Chao ◽  
J Lawler ◽  
JL Tullis
Keyword(s):  
Sodium Dodecyl ◽  
Test System ◽  
Clotting Time ◽  
Platelet Surface ◽  
Platelet Protein ◽  
Coomassie Blue ◽  
Patient Groups ◽  
Protein Alterations ◽  
Sds Electrophoresis ◽  
Tritium Labeling

Abstract Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.

scholarly journals Further characterization of HeLa S3 plasma membrane ghosts

1978 ◽  
Vol 77 (2) ◽  
pp. 448-463 ◽  
Author(s):  
E Costantino-Ceccarini ◽  
PM Novikoff ◽  
PH Atkinson ◽  
AB Novikoff
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Simple Procedure ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Major Band ◽  
Plasma Membrane Fraction ◽  
Coomassie Blue ◽  
Hela S3

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.

scholarly journals Purification and properties of β-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1079-1087 ◽  
Author(s):  
H Villarroya ◽  
J Williams ◽  
P Dey ◽  
S Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Electrophoretic Method ◽  
Germinated Seeds

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

scholarly journals Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis

2017 ◽  
Vol 5 (2) ◽  
pp. 31-38 ◽  
Author(s):  
Virginie Jakob ◽  
Livia Brunner ◽  
Christophe Barnier-Quer ◽  
Molly Blust ◽  
Nicolas Collin ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Characterization ◽  
Polyacrylamide Gel ◽  
Blue Staining ◽  
Staining Procedure ◽  
Water Emulsion ◽  
Oil In Water ◽  
Sds Page

Objectives: Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. Methods: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. Results: The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. Conclusions: These methods may be useful for improving characterization approaches of antigen–adjuvant mixtures by SDS-PAGE.

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