SNX27 Antibody (Rabbit mAb) [P13M24]

Application: Reactivity: Human, Mouse, Rat

Lane 1: K-562, Lane 2: Jurkat, Lane 3: Mouse brain, Lane 4: Rat brain

Usage Information

Dilution
WB1:500-1:1000 IP1:30 FCM1:50

Application
WB, IP, FCM

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 Observed MW
61 kDa 60 kDa,36 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	Rat spinal cord tissue; Mouse brain tissue; Mouse thymus tissue; Mouse spinal cord tissue; Rat brain tissue; Human cerebellum tissue; NIH/3T3 cells; U-87 MG cells; K-562 cells; Jurkat cells; PC-12 cells; C6 cells; EL4 cells; 3T3-L1 cells; CTLL-2 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.

Datasheet & SDS

Biological Description

Specificity
SNX27 Antibody (Rabbit mAb) [P13M24] detects endogenous levels of total SNX27 protein.

Subcellular Location
Cytoplasm, Endosome, Membrane

Uniprot ID
Q96L92

Clone
P13M24

Synonym(s)
Kiaa0488, Snx27, Sorting nexin-27

Background
SNX27 belongs to the sorting nexin family of PX domain-containing trafficking proteins and functions as a peripheral membrane cargo adaptor built from three domains arranged N- to C-terminally: a PDZ domain that selects transmembrane cargo, a central PX domain that binds the endosomal lipid phosphatidylinositol 3-phosphate, and a C-terminal FERM-like domain that engages a separate class of NPxY/NxxY-containing cargo motifs, giving SNX27 two structurally distinct cargo-recognition surfaces on a single molecule. Following endocytosis, internalized transmembrane proteins carrying a C-terminal PDZ-binding motif are delivered to early endosomes, where SNX27's PDZ domain captures this motif and diverts the cargo away from the default lysosomal degradation pathway and toward recycling back to the plasma membrane. Crystal structure and NMR analysis of the SNX27 PDZ domain bound to the retromer subunit VPS26A reveal that an exposed beta-hairpin insertion within the SNX27 PDZ domain, a feature not present in canonical PDZ domains, engages a conserved groove formed between the two lobes of VPS26A's arrestin-like fold, and this same PDZ domain surface simultaneously accommodates both the retromer-binding beta-hairpin interaction and the PDZ-binding-motif cargo interaction, structurally coupling cargo capture to retromer engagement rather than treating these as sequential, independent binding events. This coupling is functionally cooperative: VPS26A binding to the SNX27 PDZ domain increases the domain's affinity for cargo PDZ-binding motifs by roughly an order of magnitude, meaning retromer engagement actively enhances cargo selectivity rather than passively following it, and disrupting the SNX27-VPS26A binding interface abolishes cargo recycling from endosomes to the plasma membrane. Beyond the core retromer trimer of VPS26, VPS29, and VPS35, SNX27-retromer additionally couples to the WASH complex through the FAM21 subunit, which engages both retromer and SNX27 via distinct, non-overlapping motif-based contacts, and this WASH engagement drives actin polymerization that organizes the endosomal membrane into tubular subdomains from which SNX27-bound cargo is packaged into recycling carriers, so that increasing cargo concentration on the endosome membrane recruits retromer-SNX27, which in turn recruits WASH to build the tubule required for the recycling step to proceed. SNX27 also engages phosphorylated cargo through phosphorylation-dependent binding sites that substitute for the acidic residues normally required for high-affinity PDZ engagement, extending SNX27's cargo range to receptors such as the beta-2 adrenergic receptor and the NMDA receptor that lack the canonical acidic sequence motif.

Background

SNX27 belongs to the sorting nexin family of PX domain-containing trafficking proteins and functions as a peripheral membrane cargo adaptor built from three domains arranged N- to C-terminally: a PDZ domain that selects transmembrane cargo, a central PX domain that binds the endosomal lipid phosphatidylinositol 3-phosphate, and a C-terminal FERM-like domain that engages a separate class of NPxY/NxxY-containing cargo motifs, giving SNX27 two structurally distinct cargo-recognition surfaces on a single molecule. Following endocytosis, internalized transmembrane proteins carrying a C-terminal PDZ-binding motif are delivered to early endosomes, where SNX27's PDZ domain captures this motif and diverts the cargo away from the default lysosomal degradation pathway and toward recycling back to the plasma membrane. Crystal structure and NMR analysis of the SNX27 PDZ domain bound to the retromer subunit VPS26A reveal that an exposed beta-hairpin insertion within the SNX27 PDZ domain, a feature not present in canonical PDZ domains, engages a conserved groove formed between the two lobes of VPS26A's arrestin-like fold, and this same PDZ domain surface simultaneously accommodates both the retromer-binding beta-hairpin interaction and the PDZ-binding-motif cargo interaction, structurally coupling cargo capture to retromer engagement rather than treating these as sequential, independent binding events. This coupling is functionally cooperative: VPS26A binding to the SNX27 PDZ domain increases the domain's affinity for cargo PDZ-binding motifs by roughly an order of magnitude, meaning retromer engagement actively enhances cargo selectivity rather than passively following it, and disrupting the SNX27-VPS26A binding interface abolishes cargo recycling from endosomes to the plasma membrane. Beyond the core retromer trimer of VPS26, VPS29, and VPS35, SNX27-retromer additionally couples to the WASH complex through the FAM21 subunit, which engages both retromer and SNX27 via distinct, non-overlapping motif-based contacts, and this WASH engagement drives actin polymerization that organizes the endosomal membrane into tubular subdomains from which SNX27-bound cargo is packaged into recycling carriers, so that increasing cargo concentration on the endosome membrane recruits retromer-SNX27, which in turn recruits WASH to build the tubule required for the recycling step to proceed. SNX27 also engages phosphorylated cargo through phosphorylation-dependent binding sites that substitute for the acidic residues normally required for high-affinity PDZ engagement, extending SNX27's cargo range to receptors such as the beta-2 adrenergic receptor and the NMDA receptor that lack the canonical acidic sequence motif.

References
https://pubmed.ncbi.nlm.nih.gov/25136126/ https://pubmed.ncbi.nlm.nih.gov/39116126/

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%