Lin28B Antibody (Rabbit mAb) [J18A6]

Application: Reactivity: Human

Lane 1: K562, Lane 2: NCCIT

Experiment Essentials

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

Usage Information

Dilution
WB1:2000 IP1:50 IF1:100-1:1000 FCM1:150

Application
WB, IP, IF, FCM

Reactivity
Human

Source
Rabbit Monoclonal Antibody

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

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
27 kDa 30 kDa, 35 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Human testis tissue; Human placenta tissue; Human breast carcinoma tissue; HEK293 cells; HepG2 cells; NCCIT cells; K562 cells; A549 cells; JAR cells; HeLa cells
Negative Control	SW480 cells

Experimental Methods

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 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) , 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. Lyse the cells by adding 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), 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.

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 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) , 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. Lyse the cells by adding 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
Lin28B Antibody (Rabbit mAb) [J18A6] detects endogenous levels of total Lin28B protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
Q6ZN17

Clone
J18A6

Synonym(s)
CSDD2, LIN28B, Protein lin-28 homolog B, Lin-28B

Background
LIN28B is an evolutionarily conserved RNA-binding protein of the Lin28 family that contains an N‑terminal cold-shock domain and a C‑terminal pair of CCHC zinc knuckle motifs, and it functions as a post-transcriptional regulator that suppresses maturation of the tumor-suppressive let‑7 microRNA family while also binding and modulating other GGAG‑containing pre‑miRNAs to control developmental timing, stemness and oncogenic signaling. The cold-shock domain and zinc knuckles act cooperatively to recognize the 5′‑GGAG‑3′ motif in the terminal loop of pre‑let‑7 and related pre‑miRNAs, forming a stable complex that blocks Dicer processing and, in many contexts, recruits terminal uridylyltransferases such as ZCCHC11/TUT4 to catalyze 3′ uridylation and subsequent degradation of these precursors, thereby reducing mature let‑7 levels and derepressing let‑7 target mRNAs including RAS, MYC and HMGA2. LIN28B also binds primary let‑7 transcripts and can sequester pri‑let‑7 species in nucleolar compartments away from the microprocessor complex, providing an additional upstream checkpoint on let‑7 biogenesis and reinforcing low let‑7 expression in developing tissues and stem-cell populations where pluripotency and proliferation are maintained. Under normal physiological conditions, LIN28B expression is highest in embryonic and fetal contexts, including placenta, fetal liver and developing intestine, where its suppression of let‑7 supports growth, metabolic activity and delayed differentiation, establishing a LIN28B–let‑7 axis that acts as a “developmental timer” for transition from proliferative to differentiated states. In cancer, LIN28B is frequently overexpressed across a wide range of solid tumors and hematologic malignancies, and intestine-targeted expression in mouse models causes intestinal hypertrophy, crypt expansion and adenocarcinoma formation that depend on let‑7 repression, demonstrating that sustained LIN28B activity in adult tissues converts its developmental growth program into an oncogenic driver by chronically lifting let‑7-mediated brakes on cell-cycle and survival signaling. LIN28B also exerts let‑7–independent effects: it binds specific mRNAs involved in metabolism, cell-cycle regulation and epithelial–mesenchymal transition, enhancing their translation or stability, and influences cancer stem cell properties, metabolic reprogramming and immune evasion, so LIN28B integrates miRNA pathway control with direct mRNA-level regulation to coordinate multiple cancer hallmarks including stemness, proliferation, migration and therapy resistance. Expression of LIN28B is regulated by transcription factors such as MYC, which induces LIN28B to reinforce let‑7 repression and amplify MYC-driven transcriptional programs, and by noncoding RNAs that bind its 3′ UTR, creating feedback loops where LIN28B, let‑7, MYC and other miRNAs reciprocally shape each other’s levels and activities to stabilize either a stem-like oncogenic state or a more differentiated state depending on context. Collectively, these findings describe LIN28B as a cold-shock and zinc-knuckle RNA-binding protein that structurally recognizes GGAG motifs in pri‑ and pre‑miRNAs, blocks their processing and promotes uridylation-mediated decay, suppresses the let‑7 tumor-suppressor network, and in both development and tumorigenesis orchestrates a broader regulatory program over miRNA and mRNA targets that defines cell identity, growth and metastatic potential, making LIN28B a mechanistically rich target for interventions that aim to restore let‑7 function or disrupt Lin28-driven stemness in cancer.

Background

LIN28B is an evolutionarily conserved RNA-binding protein of the Lin28 family that contains an N‑terminal cold-shock domain and a C‑terminal pair of CCHC zinc knuckle motifs, and it functions as a post-transcriptional regulator that suppresses maturation of the tumor-suppressive let‑7 microRNA family while also binding and modulating other GGAG‑containing pre‑miRNAs to control developmental timing, stemness and oncogenic signaling. The cold-shock domain and zinc knuckles act cooperatively to recognize the 5′‑GGAG‑3′ motif in the terminal loop of pre‑let‑7 and related pre‑miRNAs, forming a stable complex that blocks Dicer processing and, in many contexts, recruits terminal uridylyltransferases such as ZCCHC11/TUT4 to catalyze 3′ uridylation and subsequent degradation of these precursors, thereby reducing mature let‑7 levels and derepressing let‑7 target mRNAs including RAS, MYC and HMGA2. LIN28B also binds primary let‑7 transcripts and can sequester pri‑let‑7 species in nucleolar compartments away from the microprocessor complex, providing an additional upstream checkpoint on let‑7 biogenesis and reinforcing low let‑7 expression in developing tissues and stem-cell populations where pluripotency and proliferation are maintained. Under normal physiological conditions, LIN28B expression is highest in embryonic and fetal contexts, including placenta, fetal liver and developing intestine, where its suppression of let‑7 supports growth, metabolic activity and delayed differentiation, establishing a LIN28B–let‑7 axis that acts as a “developmental timer” for transition from proliferative to differentiated states. In cancer, LIN28B is frequently overexpressed across a wide range of solid tumors and hematologic malignancies, and intestine-targeted expression in mouse models causes intestinal hypertrophy, crypt expansion and adenocarcinoma formation that depend on let‑7 repression, demonstrating that sustained LIN28B activity in adult tissues converts its developmental growth program into an oncogenic driver by chronically lifting let‑7-mediated brakes on cell-cycle and survival signaling. LIN28B also exerts let‑7–independent effects: it binds specific mRNAs involved in metabolism, cell-cycle regulation and epithelial–mesenchymal transition, enhancing their translation or stability, and influences cancer stem cell properties, metabolic reprogramming and immune evasion, so LIN28B integrates miRNA pathway control with direct mRNA-level regulation to coordinate multiple cancer hallmarks including stemness, proliferation, migration and therapy resistance. Expression of LIN28B is regulated by transcription factors such as MYC, which induces LIN28B to reinforce let‑7 repression and amplify MYC-driven transcriptional programs, and by noncoding RNAs that bind its 3′ UTR, creating feedback loops where LIN28B, let‑7, MYC and other miRNAs reciprocally shape each other’s levels and activities to stabilize either a stem-like oncogenic state or a more differentiated state depending on context. Collectively, these findings describe LIN28B as a cold-shock and zinc-knuckle RNA-binding protein that structurally recognizes GGAG motifs in pri‑ and pre‑miRNAs, blocks their processing and promotes uridylation-mediated decay, suppresses the let‑7 tumor-suppressor network, and in both development and tumorigenesis orchestrates a broader regulatory program over miRNA and mRNA targets that defines cell identity, growth and metastatic potential, making LIN28B a mechanistically rich target for interventions that aim to restore let‑7 function or disrupt Lin28-driven stemness in cancer.

References
https://pubmed.ncbi.nlm.nih.gov/24142874/ https://pubmed.ncbi.nlm.nih.gov/23420006/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%