Fiche technique

Description biologique

Spécificité	LIN28B Antibody [A15P17] reconnaît les niveaux endogènes de la protéine LIN28B totale.
Contexte	LIN28 est une protéine de liaison à l'ARN conservée qui joue un rôle clé dans le développement. Elle interagit avec les ARNm pour réguler leur traduction et leur stabilité, et elle peut également se lier aux transcrits précurseurs ou primaires de microARN (miARN) spécifiques, inhibant ainsi leur maturation. Chez les mammifères, la famille LIN28 est composée de deux gènes, LIN28A et LIN28B, tous deux impliqués dans divers processus physiologiques, y compris le développement des muscles squelettiques, la formation de la névroglie, la production de lymphocytes, le développement des cellules germinales et le métabolisme du glucose. LIN28 sert également de régulateur important de la pluripotence des cellules souches embryonnaires (ES). Lorsqu'il est combiné avec d'autres facteurs de pluripotence comme OCT4, NANOG et SOX2, LIN28A peut aider à la reprogrammation des cellules somatiques en cellules souches pluripotentes induites (iPSCs). Pendant le développement, l'expression de LIN28A et LIN28B est étroitement contrôlée et principalement restreinte aux cellules ES et aux tissus en développement. Des recherches récentes ont montré que LIN28A et LIN28B sont régulés à la hausse dans divers cancers humains et lignées cellulaires cancéreuses, coïncidant souvent avec des niveaux réduits de miARN let-7. LIN28A et LIN28B agissent comme des oncogènes en stimulant la transformation maligne, en favorisant les métastases, en modulant l'inflammation et en maintenant les populations de cellules souches cancéreuses.

Spécificité

LIN28B Antibody [A15P17] reconnaît les niveaux endogènes de la protéine LIN28B totale.

Contexte

LIN28 est une protéine de liaison à l'ARN conservée qui joue un rôle clé dans le développement. Elle interagit avec les ARNm pour réguler leur traduction et leur stabilité, et elle peut également se lier aux transcrits précurseurs ou primaires de microARN (miARN) spécifiques, inhibant ainsi leur maturation. Chez les mammifères, la famille LIN28 est composée de deux gènes, LIN28A et LIN28B, tous deux impliqués dans divers processus physiologiques, y compris le développement des muscles squelettiques, la formation de la névroglie, la production de lymphocytes, le développement des cellules germinales et le métabolisme du glucose. LIN28 sert également de régulateur important de la pluripotence des cellules souches embryonnaires (ES). Lorsqu'il est combiné avec d'autres facteurs de pluripotence comme OCT4, NANOG et SOX2, LIN28A peut aider à la reprogrammation des cellules somatiques en cellules souches pluripotentes induites (iPSCs). Pendant le développement, l'expression de LIN28A et LIN28B est étroitement contrôlée et principalement restreinte aux cellules ES et aux tissus en développement. Des recherches récentes ont montré que LIN28A et LIN28B sont régulés à la hausse dans divers cancers humains et lignées cellulaires cancéreuses, coïncidant souvent avec des niveaux réduits de miARN let-7. LIN28A et LIN28B agissent comme des oncogènes en stimulant la transformation maligne, en favorisant les métastases, en modulant l'inflammation et en maintenant les populations de cellules souches cancéreuses.

Informations dutilisation

Application

WB, IP

Dilution

WB	IP
1:1000	1:100

Réactivité

Human

Source

Rabbit Monoclonal Antibody

MW

32, 21 kDa

Tampon de stockage

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

Stockage
(À partir de la date de réception)

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

Méthodes expérimentales
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.

Méthodes expérimentales

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.

Références

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

Données dapplication

WB

Validé par Selleck

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