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Biologische Beschrijving

Specificiteit	CNBP Antibody [E10D2] detecteert endogene niveaus van totaal CNBP-eiwit.
Achtergrond	CNBP (ZNF9) is een sterk geconserveerd CCHC-type zinkvinger-nucleïnezuurbindend eiwit dat cruciaal is voor de embryonale ontwikkeling en post-transcriptionele genregulatie, gekenmerkt door zeven tandem zinkknokkelmotieven die zinkionen coördineren om compacte ββα-vouwen te vormen, waardoor een hoge-affiniteitsbinding aan G-rijke enkelstrengs DNA- en RNA-sequenties mogelijk is. De aanwezigheid van een RGG-box tussen de eerste en tweede zinkvinger vergemakkelijkt de vorming van fasegescheiden biomoleculaire druppels en verbetert de coöperatieve nucleïnezuurhermodellering. CNBP functioneert als een G-quadruplex (G4) chaperone, die actief stabiele G4-structuren binnen de c-Myc-promotor ontvouwt om RNA-polymerase II-transcriptie te bevorderen, en binnen de 5'UTR van ODC-mRNA om cap-onafhankelijke translatie te vergemakkelijken. CNBP kan genen zoals β-catenine en IL-6 transactiveren via directe, G4-afhankelijke promotorbinding, waarbij de activiteit en lokalisatie dynamisch worden gereguleerd door fosforylering-getriggerde dimerisatie en nucleaire translocatie als reactie op cellulaire stress en cytokinesignalering. Expansie van (CCTG)n-herhalingen in CNBP-intron 1 ligt ten grondslag aan myotone dystrofie type 2 (DM2), waarbij mutante RNA-transcripten functioneel CNBP sequesteren, wat resulteert in spierblind-achtige eiwittoxiciteit, verminderde myogenese als gevolg van myf5 G4-stabilisatie, en progressieve multisysteematrofie.

Specificiteit

CNBP Antibody [E10D2] detecteert endogene niveaus van totaal CNBP-eiwit.

Achtergrond

CNBP (ZNF9) is een sterk geconserveerd CCHC-type zinkvinger-nucleïnezuurbindend eiwit dat cruciaal is voor de embryonale ontwikkeling en post-transcriptionele genregulatie, gekenmerkt door zeven tandem zinkknokkelmotieven die zinkionen coördineren om compacte ββα-vouwen te vormen, waardoor een hoge-affiniteitsbinding aan G-rijke enkelstrengs DNA- en RNA-sequenties mogelijk is. De aanwezigheid van een RGG-box tussen de eerste en tweede zinkvinger vergemakkelijkt de vorming van fasegescheiden biomoleculaire druppels en verbetert de coöperatieve nucleïnezuurhermodellering. CNBP functioneert als een G-quadruplex (G4) chaperone, die actief stabiele G4-structuren binnen de c-Myc-promotor ontvouwt om RNA-polymerase II-transcriptie te bevorderen, en binnen de 5'UTR van ODC-mRNA om cap-onafhankelijke translatie te vergemakkelijken. CNBP kan genen zoals β-catenine en IL-6 transactiveren via directe, G4-afhankelijke promotorbinding, waarbij de activiteit en lokalisatie dynamisch worden gereguleerd door fosforylering-getriggerde dimerisatie en nucleaire translocatie als reactie op cellulaire stress en cytokinesignalering. Expansie van (CCTG)n-herhalingen in CNBP-intron 1 ligt ten grondslag aan myotone dystrofie type 2 (DM2), waarbij mutante RNA-transcripten functioneel CNBP sequesteren, wat resulteert in spierblind-achtige eiwittoxiciteit, verminderde myogenese als gevolg van myf5 G4-stabilisatie, en progressieve multisysteematrofie.

Gebruiksinformatie

Toepassing

WB, IP

Verdunning

WB

1:5000-1:50000

Reactiviteit

Mouse, Rat, Human

Bron

Mouse Monoclonal Antibody

MW

19 kDa

Opslagbuffer

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Opslag
(Vanaf de datum van ontvangst)

-20°C (avoid freeze-thaw cycles), 2 years

Experimentele Methoden
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:5000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimentele Methoden

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:5000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Referenties

https://pubmed.ncbi.nlm.nih.gov/31219592/
https://pubmed.ncbi.nlm.nih.gov/34474118/

Toepassingsgegevens

WB

Gevalideerd door Selleck

Lane 1: LNCAP, Lane 2: Hela, Lane 3: Jurkat, Lane 4: NIH/3T3