PABP1 Antibody (Mouse mAb) [G23P5]

Application: Reactivity: Human

Lane 1: HeLa

Usage Information

Dilution
WB1:1000 IP1:400 IF1:2000 FCM1:2000

Application
WB, IP, IF, FCM

Reactivity
Human

Source
Mouse 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
71 kDa

Positive Control	HeLa cells
Negative Control

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, 120 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 1:1000), 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 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, 120 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 1:1000), 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

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
PABP1 Antibody (Mouse mAb) [G23P5] detects endogenous levels of total PABP1 protein.

Subcellular Location
Cell projection, Cytoplasm, Nucleus, Spliceosome

Uniprot ID
P11940

Clone
G23P5

Synonym(s)
PAB1, PABP, PABP1, PABPC2, PABPC1, Polyadenylate-binding protein 1, PABP-1, Poly(A)-binding protein 1

Background
Poly(A)‑binding protein (PABP), typified by the cytoplasmic isoform PABPC1, belongs to a conserved family of RNA‑recognition motif–containing proteins that bind the poly(A) tail of eukaryotic mRNAs and act as multifunctional scaffolds for post‑transcriptional control of gene expression, integrating nuclear polyadenylation and export with cytoplasmic translation, termination and mRNA stability. The protein contains a series of N‑terminal RNA recognition motifs that contact adenylate residues within the poly(A) tail and a C‑terminal PABC domain that interacts with effector proteins such as eIF4G and PAIP1/PAIP2, creating a modular architecture that couples RNA binding to recruitment of translation factors and regulatory partners. During translation initiation, PABP bound to the 3′ poly(A) tail engages eIF4G in the eIF4F complex at the 5′ cap, promoting formation of a closed‑loop messenger ribonucleoprotein structure that enhances ribosome recruitment, supports efficient reinitiation and protects the mRNA from exonucleolytic attack, so PABP occupancy on the tail directly tunes translational output across many transcripts. At the termination step, PABP interacts with eukaryotic release factors and stimulates their recruitment and stop codon recognition, thereby stabilizing proper termination, reducing readthrough and contributing to the discrimination between normal and aberrant transcripts in processes such as nonsense‑mediated decay. In mRNA decay pathways, PABP can shield the poly(A) tail from deadenylation and inhibit nonsense‑mediated decay, maintaining stability for many messages, but in specific contexts PABPC1 recruits deadenylase complexes to initiate shortening of the poly(A) tail and trigger turnover, so PABP activity on individual transcripts depends on its engagement with partner RNA‑binding proteins and decay enzymes. Nuclear forms of PABP contribute to poly(A) tail synthesis by regulating length during polyadenylation and facilitating maturation and export of newly formed mRNPs, connecting co‑transcriptional RNA processing with downstream cytoplasmic control by the same protein family. Developmental studies show that distinct PABP family members have non‑redundant roles: cytoplasmic PABPs and embryonic PABP (EPABP) regulate maternal mRNA translation in vertebrate oocytes, and loss of EPABP causes infertility with oocyte maturation defects, indicating that PABP‑mediated control of poly(A)‑dependent translation is essential for early developmental programs. In neurodegenerative disease, PABP1 localizes to a subset of cytoplasmic TDP‑43 inclusions and stress granules in amyotrophic lateral sclerosis, particularly in patients with C9orf72 expansions, consistent with its role as a core stress granule component and its interaction with RNA‑binding proteins linked to ALS, pointing to perturbation of PABP‑centered mRNP assemblies in motor neuron pathology.

Background

Poly(A)‑binding protein (PABP), typified by the cytoplasmic isoform PABPC1, belongs to a conserved family of RNA‑recognition motif–containing proteins that bind the poly(A) tail of eukaryotic mRNAs and act as multifunctional scaffolds for post‑transcriptional control of gene expression, integrating nuclear polyadenylation and export with cytoplasmic translation, termination and mRNA stability. The protein contains a series of N‑terminal RNA recognition motifs that contact adenylate residues within the poly(A) tail and a C‑terminal PABC domain that interacts with effector proteins such as eIF4G and PAIP1/PAIP2, creating a modular architecture that couples RNA binding to recruitment of translation factors and regulatory partners. During translation initiation, PABP bound to the 3′ poly(A) tail engages eIF4G in the eIF4F complex at the 5′ cap, promoting formation of a closed‑loop messenger ribonucleoprotein structure that enhances ribosome recruitment, supports efficient reinitiation and protects the mRNA from exonucleolytic attack, so PABP occupancy on the tail directly tunes translational output across many transcripts. At the termination step, PABP interacts with eukaryotic release factors and stimulates their recruitment and stop codon recognition, thereby stabilizing proper termination, reducing readthrough and contributing to the discrimination between normal and aberrant transcripts in processes such as nonsense‑mediated decay. In mRNA decay pathways, PABP can shield the poly(A) tail from deadenylation and inhibit nonsense‑mediated decay, maintaining stability for many messages, but in specific contexts PABPC1 recruits deadenylase complexes to initiate shortening of the poly(A) tail and trigger turnover, so PABP activity on individual transcripts depends on its engagement with partner RNA‑binding proteins and decay enzymes. Nuclear forms of PABP contribute to poly(A) tail synthesis by regulating length during polyadenylation and facilitating maturation and export of newly formed mRNPs, connecting co‑transcriptional RNA processing with downstream cytoplasmic control by the same protein family. Developmental studies show that distinct PABP family members have non‑redundant roles: cytoplasmic PABPs and embryonic PABP (EPABP) regulate maternal mRNA translation in vertebrate oocytes, and loss of EPABP causes infertility with oocyte maturation defects, indicating that PABP‑mediated control of poly(A)‑dependent translation is essential for early developmental programs. In neurodegenerative disease, PABP1 localizes to a subset of cytoplasmic TDP‑43 inclusions and stress granules in amyotrophic lateral sclerosis, particularly in patients with C9orf72 expansions, consistent with its role as a core stress granule component and its interaction with RNA‑binding proteins linked to ALS, pointing to perturbation of PABP‑centered mRNP assemblies in motor neuron pathology.

References
https://pubmed.ncbi.nlm.nih.gov/12844354/ https://pubmed.ncbi.nlm.nih.gov/27418677/

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%