scholarly journals ACTB and GAPDH proteins appear at multiple positions of SDS-PAGE and may not be suitable for serving as reference genes for the protein level determination in such techniques as Western blotting

2020 ◽  
Author(s):  
Yan He ◽  
Ju Zhang ◽  
Jiayuan Qu ◽  
Lucas Zellmer ◽  
Yan Zhao ◽  
...  
Keyword(s):  
Western Blotting ◽  
Protein Level ◽  
Reference Genes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioinformatic Analysis ◽  
Protein Isoforms ◽  
Leading Role ◽  
Non Coding Rna ◽  
Sds Page

AbstractMost human genes can produce multiple protein isoforms that should appear at multiple positions of polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS), but most published results of Western blotting show only one protein. We performed SDS-PAGE of proteins from several human cell lines, isolated the proteins at the 72-, 55-, 48-, 40-, and 26-kD positions, and used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although ACTB and GAPDH are 41.7-kD and 36-kD proteins, respectively, LC-MS/MS identified peptides of ACTB and GAPDH at all of these SDS-PAGE positions, making us wonder whether they produce some unknown protein isoforms. The NCBI (National Center for Biotechnology Information, USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one non-coding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatic analysis identified a 17.6-kD isoform encoded by the non-coding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with great similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to ACTB. These data collectively disqualify GAPDH and ACTB from serving as the reference genes for determination of the protein level in such techniques as Western blotting, a leading role these two genes have been playing for decades in the biomedical research.

scholarly journals ACTB and GAPDH appear at multiple SDS-PAGE positions, thus not suitable as reference genes for determining protein loading in techniques like Western blotting

2021 ◽  
Vol 16 (1) ◽  
pp. 1278-1292
Author(s):  
Keyin Zhang ◽  
Ju Zhang ◽  
Nan Ding ◽  
Lucas Zellmer ◽  
Yan Zhao ◽  
...  
Keyword(s):  
Western Blotting ◽  
Reference Genes ◽  
Noncoding Rna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Isoforms ◽  
Protein Loading ◽  
Leading Role ◽  
C Terminus ◽  
Human Proteins

Abstract We performed polyacrylamide gel electrophoresis of human proteins with sodium dodecyl sulfate, isolated proteins at multiple positions, and then used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are 41.7 and 36 kDa proteins, respectively, LC-MS/MS identified their peptides at all the positions studied. The National Center for Biotechnology Information (USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one noncoding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatics analysis identified a 17.6 kDa isoform encoded by the noncoding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with significant similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to the ACTB. These data lead us to conclude that GAPDH and ACTB are poor reference genes for determining the protein loading in such techniques as Western blotting, a leading role these two genes have been playing for decades in biomedical research.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

2007 ◽  
Vol 53 (4) ◽  
pp. 526-532 ◽  
Author(s):  
Priyanka D. Abeyrathne ◽  
Joseph S. Lam
Keyword(s):  
Membrane Proteins ◽  
Western Blotting ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bacterial Membrane ◽  
Nitrocellulose Membrane ◽  
Western Blots ◽  
Sds Page ◽  
Highly Hydrophobic ◽  
Effective Transfer

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS–PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2–12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

scholarly journals Identification and Purification of SpecificPenicillium marneffei Antigens and Their Recognition by Human Immune Sera

1998 ◽  
Vol 36 (4) ◽  
pp. 949-954 ◽  
Author(s):  
L. Jeavons ◽  
A. J. Hamilton ◽  
N. Vanittanakom ◽  
R. Ungpakorn ◽  
E. G. V. Evans ◽  
...  
Keyword(s):  
Western Blotting ◽  
Gel Electrophoresis ◽  
Pathogenic Fungus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Sds Page ◽  
Molecular Masses

Disseminated infection with the dimorphic pathogenic fungusPenicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.

scholarly journals Brown Snakes (Pseudonaja Genus): Venom Yields, Prothrombin Activator Neutralization and Implications Affecting Antivenom Usage

1998 ◽  
Vol 26 (3) ◽  
pp. 276-281 ◽  
Author(s):  
P. P. Masci ◽  
P. J. Mirtschin ◽  
T. N. Nias ◽  
R. K. Turnbull ◽  
T. R. Kuchel ◽  
...  
Keyword(s):  
Western Blotting ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Time Dependent ◽  
Adult Rats ◽  
Sds Page ◽  
High Prevalence ◽  
The Common ◽  
First Time

The recent high prevalence of fatal bites by Brown snakes (Pseudonaja genus) has led to this study of venom yields from 66 brown snake milkings over 15 months. The amount of venom obtained from all species was higher than reported previously. Electrophoretic and Western blotting analyses of their venoms showed significantly lower avidity of Brown snake antivenom (BS-AV) for the prothrombin activator (PA) component (190 kD) than for other venom components, including the neurotoxins. The LD50 of P. inframacula has been determined for the first time. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and Western blotting studies have shown that the Pseudonaja venoms contained proportionately more PA component than venoms of the Taipan (Oxyuranus scutellatus) or the Fierce snake (O. microlepidotus). Neutralization of the prothrombin activator of the Common Brown snake (P. textilis) (Pt-PA) by BS-AV was found to be time dependent and 40% remained unneutralized after 30 minutes incubation. Adult rats administered quantities of Pt-PA (IV) died with acute disseminated intravascular coagulation. Rats were made resistant to Pt-PA by preheparinization or by induction of tolerance to increasing quantities of Pt-PA. There is no evidence that Pt-PA has intrinsic toxicity apart from being a procoagulant. The improvement of BS-AV by addressing its deficiencies should be canvassed.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

2020 ◽  
Vol 20 (8) ◽  
pp. 970-981
Author(s):  
Hamed A. Ghramh ◽  
Essam H. Ibrahim ◽  
Mona Kilnay
Keyword(s):  
Anticancer Activity ◽  
Cancer Cells ◽  
Biological Activities ◽  
Sugar Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioactive Molecules ◽  
Sds Page ◽  
Splenic Cells ◽  
Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

scholarly journals Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Preeti Anand ◽  
Jay Prakash Pandey ◽  
Dev Mani Pandey
Keyword(s):  
San Francisco ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Evolutionary Genetics ◽  
Imaging Analysis ◽  
Silk Cocoon ◽  
Natural Color ◽  
Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

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