Datenblatt

Biologische Beschreibung

Spezifität	LIN28B Antibody [A15P17] erkennt endogene Spiegel des gesamten LIN28B-Proteins.
Hintergrund	LIN28 ist ein konserviertes RNA-bindendes Protein, das eine Schlüsselrolle in der Entwicklung spielt. Es interagiert mit mRNAs, um deren Translation und Stabilität zu regulieren, und es kann auch an Vorläufer- oder Primärtranskripte spezifischer microRNAs (miRNAs) binden, wodurch deren Reifung gehemmt wird. Bei Säugetieren besteht die LIN28-Familie aus zwei Genen, LIN28A und LIN28B, die beide an verschiedenen physiologischen Prozessen beteiligt sind, einschließlich der Entwicklung der Skelettmuskulatur, der Neuroglia-Bildung, der Lymphozytenproduktion, der Keimzellentwicklung und des Glukosestoffwechsels. LIN28 dient auch als wichtiger Regulator der Pluripotenz embryonaler Stammzellen (ES-Zellen). In Kombination mit anderen Pluripotenzfaktoren wie OCT4, NANOG und SOX2 kann LIN28A bei der Reprogrammierung somatischer Zellen in induzierte pluripotente Stammzellen (iPSCs) helfen. Während der Entwicklung wird die Expression von LIN28A und LIN28B streng kontrolliert und ist hauptsächlich auf ES-Zellen und sich entwickelnde Gewebe beschränkt. Jüngste Forschungen haben ergeben, dass LIN28A und LIN28B in verschiedenen menschlichen Krebsarten und Krebszelllinien hochreguliert sind, oft einhergehend mit reduzierten Spiegeln von let-7 miRNAs. LIN28A und LIN28B fungieren als Onkogene, indem sie die maligne Transformation vorantreiben, die Metastasierung fördern, Entzündungen modulieren und Krebsstammzellpopulationen aufrechterhalten.

Spezifität

LIN28B Antibody [A15P17] erkennt endogene Spiegel des gesamten LIN28B-Proteins.

Hintergrund

LIN28 ist ein konserviertes RNA-bindendes Protein, das eine Schlüsselrolle in der Entwicklung spielt. Es interagiert mit mRNAs, um deren Translation und Stabilität zu regulieren, und es kann auch an Vorläufer- oder Primärtranskripte spezifischer microRNAs (miRNAs) binden, wodurch deren Reifung gehemmt wird. Bei Säugetieren besteht die LIN28-Familie aus zwei Genen, LIN28A und LIN28B, die beide an verschiedenen physiologischen Prozessen beteiligt sind, einschließlich der Entwicklung der Skelettmuskulatur, der Neuroglia-Bildung, der Lymphozytenproduktion, der Keimzellentwicklung und des Glukosestoffwechsels. LIN28 dient auch als wichtiger Regulator der Pluripotenz embryonaler Stammzellen (ES-Zellen). In Kombination mit anderen Pluripotenzfaktoren wie OCT4, NANOG und SOX2 kann LIN28A bei der Reprogrammierung somatischer Zellen in induzierte pluripotente Stammzellen (iPSCs) helfen. Während der Entwicklung wird die Expression von LIN28A und LIN28B streng kontrolliert und ist hauptsächlich auf ES-Zellen und sich entwickelnde Gewebe beschränkt. Jüngste Forschungen haben ergeben, dass LIN28A und LIN28B in verschiedenen menschlichen Krebsarten und Krebszelllinien hochreguliert sind, oft einhergehend mit reduzierten Spiegeln von let-7 miRNAs. LIN28A und LIN28B fungieren als Onkogene, indem sie die maligne Transformation vorantreiben, die Metastasierung fördern, Entzündungen modulieren und Krebsstammzellpopulationen aufrechterhalten.

Nutzungsinformationen

Anwendung

WB, IP

Verdünnung

WB	IP
1:1000	1:100

Reaktivität

Human

Quelle

Rabbit Monoclonal Antibody

MW

32, 21 kDa

Lagerpuffer

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

Lagerung
(Ab dem Datum des Erhalts)

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

Experimentelle 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 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. ( 0.45 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 1:1000), 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 1048. 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.

Experimentelle 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

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. ( 0.45 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 1:1000), 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

1048. 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.

Referenzen

https://pubmed.ncbi.nlm.nih.gov/30174831/

Anwendungsdaten

WB

Validiert von Selleck

Lane 1: NTERA-2
Lane 2: Hep G2
Lane 3: Huh7