PEX5 Antibody [L22A16] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: Raji, Lane 2: Jurkat, Lane 3: Hela, Lane 4: Ramos

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat

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
80 kDa

Positive Control	Raji cells; Jurkat cells; HeLa cells; Ramos cells; BaF3 cells; RBL-2H3 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. 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. 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 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 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.

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.

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 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

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.

Datasheet & SDS

Biological Description

Specificity
PEX5 Antibody [L22A16] detects endogenous levels of total PEX5 protein.

Subcellular Location
Cytoplasm, Peroxisome

Uniprot ID
P50542

Clone
L22A16

Synonym(s)
Peroxisomal targeting signal 1 receptor; PTS1 receptor; PTS1R; PTS1-BP; Peroxin-5; Peroxisomal C-terminal targeting signal import receptor; Peroxisome receptor 1; PEX5; PXR1

Background
PEX5 is the primary cytosolic receptor for peroxisomal targeting signal 1 (PTS1)-containing matrix proteins among the peroxin family, which is essential for organelle biogenesis. The protein features an intrinsically disordered N-terminal domain with pentapeptide motifs and amphipathic helices that interact with docking peroxins PEX14 and PEX13. Its C-terminal tetratricopeptide repeat (TPR) domain forms a six-helix bundle responsible for recognizing the C-terminal PTS1 tripeptide of cargo proteins such as catalase. In the cytosol, PEX5 captures folded PTS1 cargos and, via its longer isoform PEX5L, binds the PEX7/PTS2 complex. It then docks at the peroxisomal membrane through interactions with PEX14. Cargo translocation into the matrix occurs as PEX5 inserts into the membrane, adopting a transmembrane topology with its N-terminus exposed to the cytosol. Here, PEX5 is monoubiquitinated at a conserved cysteine by PEX4/PEX22 and the E3 ligase PEX12. This ubiquitin tag initiates extraction by the receptor export module, PEX1/PEX6 AAA ATPases and PEX26, which unfold PEX5 and releases the cargo into the peroxisome matrix. Monoubiquitinated PEX5 binds more tightly to export components than its unmodified form, ensuring rapid recycling and continuous import cycles. This process is critical for maintaining peroxisome dynamics, lipid metabolism, and reactive oxygen species (ROS) detoxification by sustaining matrix enzyme levels. PEX5 also participates in the import of PTS2 proteins indirectly by tethering PEX7, thus coordinating dual targeting pathways. Dysfunction involving PEX5 ubiquitination or domain mutations impairs import and leads to peroxisome biogenesis disorders such as Zellweger syndrome, characterized by severe neurological defects, hypotonia, and early lethality due to enzyme deficiencies. Altered PEX5 cycling affects ROS signaling and tumor progression through impaired peroxisomal beta-oxidation.

Background

PEX5 is the primary cytosolic receptor for peroxisomal targeting signal 1 (PTS1)-containing matrix proteins among the peroxin family, which is essential for organelle biogenesis. The protein features an intrinsically disordered N-terminal domain with pentapeptide motifs and amphipathic helices that interact with docking peroxins PEX14 and PEX13. Its C-terminal tetratricopeptide repeat (TPR) domain forms a six-helix bundle responsible for recognizing the C-terminal PTS1 tripeptide of cargo proteins such as catalase. In the cytosol, PEX5 captures folded PTS1 cargos and, via its longer isoform PEX5L, binds the PEX7/PTS2 complex. It then docks at the peroxisomal membrane through interactions with PEX14. Cargo translocation into the matrix occurs as PEX5 inserts into the membrane, adopting a transmembrane topology with its N-terminus exposed to the cytosol. Here, PEX5 is monoubiquitinated at a conserved cysteine by PEX4/PEX22 and the E3 ligase PEX12. This ubiquitin tag initiates extraction by the receptor export module, PEX1/PEX6 AAA ATPases and PEX26, which unfold PEX5 and releases the cargo into the peroxisome matrix. Monoubiquitinated PEX5 binds more tightly to export components than its unmodified form, ensuring rapid recycling and continuous import cycles. This process is critical for maintaining peroxisome dynamics, lipid metabolism, and reactive oxygen species (ROS) detoxification by sustaining matrix enzyme levels. PEX5 also participates in the import of PTS2 proteins indirectly by tethering PEX7, thus coordinating dual targeting pathways. Dysfunction involving PEX5 ubiquitination or domain mutations impairs import and leads to peroxisome biogenesis disorders such as Zellweger syndrome, characterized by severe neurological defects, hypotonia, and early lethality due to enzyme deficiencies. Altered PEX5 cycling affects ROS signaling and tumor progression through impaired peroxisomal beta-oxidation.

References
https://pubmed.ncbi.nlm.nih.gov/28933989/ https://pubmed.ncbi.nlm.nih.gov/35931083/

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%