TRIM5α Antibody [E3H20] Datasheet

Biological Description

Specificity	TRIM5α Antibody [E3H20] detects endogenous levels of total TRIM5α protein.
Background	TRIM5α belongs to the tripartite motif (TRIM) family of proteins characterized by an N-terminal RING domain with E3 ubiquitin ligase activity, B-box domains, and a coiled-coil region that mediates oligomerization, followed by a C-terminal B30.2/SPRY domain responsible for specific ligand recognition. The coiled-coil domain enables higher-order assembly into hexagonal lattices that match the symmetry of retroviral capsid surfaces, while the SPRY domain directly engages determinants on the viral capsid lattice in a species-variable manner, with rhesus monkey TRIM5α showing high avidity for HIV-1 capsid and human TRIM5α preferring N-tropic murine leukemia virus capsid. Upon binding the intact incoming retroviral core via the SPRY domain, TRIM5α accelerates premature uncoating, converting the particulate capsid into soluble forms in the cytosol without relying on proteasomal degradation or major covalent modifications to the capsid protein, thereby disrupting early post-entry events prior to reverse transcription. Concurrently, TRIM5α partners with the UBC13/UEV1A heterodimeric E2 ubiquitin-conjugating enzyme to synthesize free, unattached K63-linked polyubiquitin chains through its RING domain, which bind TAB2/3 and trigger autophosphorylation of the TAK1 kinase complex at threonine 187, leading to downstream activation of AP-1 and NF-κB transcription factors that drive inflammatory cytokine and chemokine expression. This dual mechanism integrates retroviral restriction with innate immune sensing, where capsid lattice interaction amplifies ubiquitin chain production and signaling potency scales with TRIM5α binding avidity to the capsid, enhancing production of mediators like IL-6, IL-8, CXCL10, and PTGS2 upon challenge by restriction-sensitive retroviruses. TRIM5α operates in myeloid cells and restricts diverse retroviruses, including HIV-1 in Old World monkeys and MLV strains in humans, contributing to species-specific viral tropism and bolstering antiviral states induced by pathogen-associated patterns like LPS through TAK1-dependent pathways.

Specificity

TRIM5α Antibody [E3H20] detects endogenous levels of total TRIM5α protein.

Background

TRIM5α belongs to the tripartite motif (TRIM) family of proteins characterized by an N-terminal RING domain with E3 ubiquitin ligase activity, B-box domains, and a coiled-coil region that mediates oligomerization, followed by a C-terminal B30.2/SPRY domain responsible for specific ligand recognition. The coiled-coil domain enables higher-order assembly into hexagonal lattices that match the symmetry of retroviral capsid surfaces, while the SPRY domain directly engages determinants on the viral capsid lattice in a species-variable manner, with rhesus monkey TRIM5α showing high avidity for HIV-1 capsid and human TRIM5α preferring N-tropic murine leukemia virus capsid. Upon binding the intact incoming retroviral core via the SPRY domain, TRIM5α accelerates premature uncoating, converting the particulate capsid into soluble forms in the cytosol without relying on proteasomal degradation or major covalent modifications to the capsid protein, thereby disrupting early post-entry events prior to reverse transcription. Concurrently, TRIM5α partners with the UBC13/UEV1A heterodimeric E2 ubiquitin-conjugating enzyme to synthesize free, unattached K63-linked polyubiquitin chains through its RING domain, which bind TAB2/3 and trigger autophosphorylation of the TAK1 kinase complex at threonine 187, leading to downstream activation of AP-1 and NF-κB transcription factors that drive inflammatory cytokine and chemokine expression. This dual mechanism integrates retroviral restriction with innate immune sensing, where capsid lattice interaction amplifies ubiquitin chain production and signaling potency scales with TRIM5α binding avidity to the capsid, enhancing production of mediators like IL-6, IL-8, CXCL10, and PTGS2 upon challenge by restriction-sensitive retroviruses. TRIM5α operates in myeloid cells and restricts diverse retroviruses, including HIV-1 in Old World monkeys and MLV strains in humans, contributing to species-specific viral tropism and bolstering antiviral states induced by pathogen-associated patterns like LPS through TAK1-dependent pathways.

Usage Information

Application

WB, IP

Dilution

WB	IP
1:1000	1:50

Reactivity

Human, Monkey

Source

Rabbit Monoclonal Antibody

MW

56 kDa

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

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. 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 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.

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.

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 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.

References

https://pubmed.ncbi.nlm.nih.gov/21512573/
https://pubmed.ncbi.nlm.nih.gov/16540544/

Application Data

WB

Validated by Selleck

Lane 1: Jurkat, Lane 2: HepG2, Lane 3: MCF7, Lane 4: COS-7