Name: Geoptimaliseerde analyse van eiwitten uit xenopus-eicellen en embryos door immunoblotting (Video) | JoVE Journal
Uploaded: 2025-09-19T14:00:00.000+00:00
Duration: 9 min 32 s
Description: Leer een geoptimaliseerd immunoblotting-protocol voor eiwitanalyse in Xenopus-oöcyten en embryo's. Deze methode maakt detectie van translationele regulatoren mogelijk voor ontwikkelingsbiologisch onderzoek.

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<table><tbody><tr><td>100 sq ft Heavy Duty Premium Wrap</td><td>Saran</td><td>2570000130</td><td></td></tr><tr><td>10x Cell Lysis Buffer</td><td>Cell Signaling Technology</td><td>#9803</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>2-Quart Oblong Baking/Serving Dish</td><td>Pyrex</td><td>71160010116</td><td></td></tr><tr><td>2x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>#1610737</td><td></td></tr><tr><td>Anti-HA High Affinity Antibody</td><td>Roche</td><td>11867423001</td><td>Prepared as directed and used at 1:10,000 dilution.</td></tr><tr><td>Anti-Rabbit IgG, HRP-Linked Antibody</td><td>Cell Signaling Technology</td><td>7074S</td><td>Used at 1:30,000 dilution.</td></tr><tr><td>Autoradiography Cassette, 5 x 7 Inches</td><td>Research Products International</td><td>420057</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>Bel-Art Disposable Pestles</td><td>Millipore Sigma</td><td>BAF199230001</td><td></td></tr><tr><td>Bicaudal-C (Human) Rabbit Antibody</td><td>Envigo Bioproducts (now Inotiv)</td><td>N/A</td><td>Custom ordered. Used at 1:20,000 dilution.</td></tr><tr><td>Bicaudal-C (Xenopus) Rabbit Antibody</td><td>Envigo Bioproducts (now Inotiv)</td><td>N/A</td><td>Custom ordered. Used at 1:20,000 dilution. Ref. Park et al., 2016</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A3294</td><td>A substitute for nonfat milk in blocking buffer.</td></tr><tr><td>Centrifuge 5425</td><td>Eppendorf</td><td>13-864-456</td><td></td></tr><tr><td>CL-XPosure Film</td><td>ThermoFisher Scientific</td><td>34091</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>CNOT1 (D5M1K) Rabbit Monoclonal Antibody</td><td>Cell Signaling Technology</td><td>#44613</td><td>Used at 1:2,000 dilution.</td></tr><tr><td>DDX6/RCK (D26C11) Rabbit Monoclonal Antibody</td><td>Cell Signaling Technology</td><td>#8988</td><td>Used at 1:5,000 dilution.</td></tr><tr><td>E-Z Store and Pour Developer</td><td>MXR Imaging</td><td>103633</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>E-Z Store and Pour Fixer</td><td>MXR Imaging</td><td>114511</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>GAPDH Monoclonal Antibody</td><td>ProteinTech</td><td>60004-1-Ig</td><td>Used at 1:40,000 dilution.</td></tr><tr><td>Gel Releaser</td><td>Bio-Rad</td><td>#1653320</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP</td><td>Invitrogen</td><td>A16066</td><td>Prepared as directed and used at 1:40,000 dilution.</td></tr><tr><td>Ice Pack, 8 oz, 5 x 3 x 1"</td><td>Uline</td><td>S-18256</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Aldrich</td><td>C7352</td><td></td></tr><tr><td>Medical Film Processor</td><td>Konica Minolta</td><td>SRX-101A</td><td>Not necessary if using a digital imaging system.</td></tr><tr><td>Mini-PROTEAN&nbsp;Cassette Opening Lever</td><td>Bio-Rad</td><td>#4560000</td><td></td></tr><tr><td>Mini-PROTEAN&nbsp;Tetra Vertical Electrophoresis Cell</td><td>Bio-Rad</td><td>#1658004</td><td></td></tr><tr><td>MSE PRO 700ml Western Blot Electrophoresis Tank</td><td>MSE Supplies</td><td>BB2369</td><td></td></tr><tr><td>Nitrocellulose Membrane, 0.45 &micro;m</td><td>Bio-Rad</td><td>#1620115</td><td></td></tr><tr><td>Nonfat Dry Milk</td><td>Sanalac</td><td>1570000180</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Rat IgG (H+L)</td><td>Jackson Immunoresearch</td><td>112-035-003</td><td>Prepared as directed and used at 1:100,000 dilution.</td></tr><tr><td>Polysorbate (Tween) 20</td><td>Fisher Bioreagents</td><td>BP337-500</td><td></td></tr><tr><td>Ponceau S</td><td>VWR Life Science</td><td>K793-500ML</td><td>An approximation can be made with 0.5% Ponceau S, 1% acetic acid</td></tr><tr><td>PowerPac Basic Power Supply</td><td>Bio-Rad</td><td>1645050</td><td></td></tr><tr><td>PR1MA Variable Speed 2D Rocker</td><td>MidSci</td><td>R2D-30</td><td></td></tr><tr><td>Precision Plus Protein Dual Color Standards Ladder</td><td>Bio-Rad</td><td>#1610374</td><td>Any protein ladder of choice can be used.</td></tr><tr><td>Pure Cellulose Chromatography Filter Paper, 0.35 mm</td><td>Fisher Scientific</td><td>05-714-4</td><td></td></tr><tr><td>Restore Western Blot Stripping Buffer</td><td>ThermoFisher Scientific</td><td>#21059</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Chemical</td><td>S640-10</td><td></td></tr><tr><td>Storage Box 300 mL</td><td>Rosti Mepal</td><td>RST46608</td><td>For nitrocellulose membrane blocking, antibody incubation, and washes</td></tr><tr><td>SuperSignal West Femto Maximum Sensitivity Substrate</td><td>ThermoFisher Scientific</td><td>#34094</td><td>An enhanced chemiluminescent (ECL) substrate containing two components: luminol and enhancer. This ECL reagent has a low-femtogram sensitivity.</td></tr><tr><td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td><td>ThermoFisher Scientific</td><td>#34580</td><td>An enhanced chemiluminescent (ECL) substrate containing two components: luminol and enhancer. This ECL reagent has a picogram to femtogram sensitivity.</td></tr><tr><td>SurePAGE, Bis-Tris, 10 x 8, 4&ndash;12%, 10 Well Polyacrylamide Gels</td><td>GenScript</td><td>M00652</td><td></td></tr><tr><td>Transfer Buffer Powder</td><td>GenScript</td><td>M00652</td><td></td></tr><tr><td>Tris Base Ultrapure</td><td>USBiological</td><td>77-86-1</td><td></td></tr><tr><td>Tris-MOPS-SDS Running Buffer Powder</td><td>GenScript</td><td>M00138</td><td></td></tr></tbody></table>

geoptimaliseerde analyse van eiwitten uit xenopus-eicellen en embryo's door immunoblotting

Optimized Analysis of Proteins from Xenopus Oocytes and Embryos by Immunoblotting

Geoptimaliseerde analyse van eiwitten uit xenopus-eicellen en embryo's door immunoblotting

Analysis of proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting (western blotting) is a vital ...

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Research

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Biochemistry

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Immunoblotting (western blotting) is een snelle en gevoelige test voor de detectie en karakterisering van eiwitten die werkt door het benutten van de specificiteit die inherent zijn aan antigeen-antilichaam erkenning. Deze video bevat protocollen voor de scheiding van eiwitten, blotting eiwitten op membranen, immunoprobing, en visualisatie met behulp van chromogene of chemiluminescent substraten.

Immunoblot analyse

Een gemeenschappelijke eiwitextractie protocol met behulp van ureum / thioureum / SDS buffer voor menselijke en muizen hersenweefsel maakt het identificeren van eiwitten door 2D-DIGE en de daaropvolgende karakterisering door mini 2DE immunoblotting. Deze methode maakt een meer reproduceerbare en betrouwbare resultaten te verkrijgen van menselijke biopsies en experimentele modellen.

Consensus Brain-afgeleide eiwit, Extraction Protocol voor de studie van mensen en muizen Brain Proteome behulp Zowel 2D-DIGE en Mini 2DE Immunoblotting

De vraag hoe chromatine toezichthouders en chromatine staten van invloed op het genoom in vivo is de sleutel tot ons begrip van hoe vroeg lot van de cel beslissingen worden genomen in het zich ontwikkelende embryo. ChIP-Seq-de meest populaire benadering van chromatine functies onderzoeken op mondiaal niveau wordt hier geschetst voor Xenopus embryo.

Genoom-brede Snapshot van chromatine regelgevers en staten in<em&gt; Xenopus</em&gt; Embryo&#39;s van Chip-Seq

Controle van eiwitsynthese vindt voornamelijk plaats tijdens de initiatiefase van translatie, waarvan tekortkomingen zijn gekoppeld aan verschillende aandoeningen. Om de etiologie beter te begrijpen, beschreven we hier een protocol met Xenopus laevis eicellen dat de translatie van het mos-transcript beoordeelt in aanwezigheid van een mutant van translatie-initiatiefactor eIF4G1.

Xenopus laevis als een model te identificeren Vertaling Impairment

Dit protocol geeft een werkwijze kwantitatieve eencellige voeren in situ analyse van eiwitexpressie van afstamming specificatie mouse pre-implantatie embryo bestuderen. De noodzakelijke voor het verzamelen van blastocysten procedures, whole-mount immunofluorescentie detectie van eiwitten, beeldvorming van de monsters op een confocale microscoop, en nucleaire segmentatie en beeldanalyse worden beschreven.

Kwantitatieve Analyse van eiwitexpressie aan Lineage Specificatie studie in muizenmodellen preimplantatie embryo&#39;s

Een nieuwe techniek voor de detectie van lage abundantie endogeen receptoren aanwezig in zebravis embryo's wordt beschreven. We hebben het AFLIP genoemd omdat het bestaat uit affiniteitslabeling van de receptor door zijn ligand gekoppeld aan immunoprecipitatie.

Affiniteit Labeling Detectie van endogene receptoren van zebravis embryo&#39;s

Deze methode beschrijft een robuuste en reproduceerbare aanpak voor het vergelijken van eiwitniveaus in verschillende weefsels en bij verschillende ontwikkelings timepoints met behulp van een kwantitatieve westelijke bevlekkende standaardbenadering.

Robuuste vergelijking van eiwitniveaus over weefsels en in de gehele ontwikkeling met behulp van gestandaardiseerde kwantitatieve westelijke bevlekken

Dit protocol beschrijft de cryoAPEX-methode, waarbij een APEX2-gelabeld membraaneiwit kan worden gelokaliseerd door transmissieelektronenmicroscopie binnen een optimaal bewaarde celultrastructuur.

De CryoAPEX-methode voor elektronenmicroscopieanalyse van membraaneiwitlokalisatie binnen ultrastructureel bewaarde cellen

Hier beschrijven we een op massaspectrometrie gebaseerde proteomische karakterisering van cellijnen met bekende weefsellotgevallen in het gewervelde Xenopus laevis-embryo .

Cel-Lineage geleide massaspectrometrie proteomics in het zich ontwikkelende (kikker) embryo

Zebravis gericht op reactieve elektrofielen en oxidanten (Z-REX) is een op chemische biologie gebaseerde methode voor het onderzoek van reactieve signalering van kleine moleculen. Deze techniek kan worden toegepast op levende vissen van verschillende ontwikkelingsstadia. Hier koppelen we standaardtests in zebravissen aan Z-REX voor signaalrouteanalyse.

Monitoring van on-target signaalreacties bij larvale zebravissen - Z-REX ontmaskert precieze mechanismen van elektrofiele geneesmiddelen en metabolieten

Leer een geoptimaliseerd immunoblotting-protocol voor eiwitanalyse in Xenopus-oöcyten en embryo's. Deze methode maakt detectie van translationele regulatoren mogelijk voor ontwikkelingsbiologisch onderzoek.

Geoptimaliseerde analyse van eiwitten uit xenopus-eicellen en embryo's door immunoblotting

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