OS9 Antibody [M17H4] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat, Monkey

Lane 1: HL60, Lane 2: MCF7, Lane 3: PANC1, Lane 4: COS-7

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Reactivity
Human, Mouse, Rat, Monkey

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
83 kDa,97 kDa

Positive Control	HeLa cells (thapsigargin, 2 nM, 16 h); SJSA‑1 cells; RH30 cells; HL‑60 cells; A‑431 cells; SN‑12C cells; MCF7 cells; ACHN cells; PANC‑1 cells; COLO 205 cells; COS‑7 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. 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.

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.

Datasheet & SDS

Biological Description

Specificity
OS9 Antibody [M17H4] detects endogenous levels of total OS9 protein.

Subcellular Location
Endoplasmic reticulum

Uniprot ID
Q13438

Clone
M17H4

Synonym(s)
Protein OS-9; ER-retained protein; OS9

Background
OS9 functions as an endoplasmic reticulum resident lectin within the mannose-6-phosphate receptor homology domain family, recognizing monoglucosylated N-glycans on misfolded glycoproteins to facilitate ER-associated degradation. The protein contains an N-terminal signal sequence, a central MRH domain with mannose-binding sites flanked by conserved charged residues, and C-terminal transmembrane and cytoplasmic tail regions that anchor it to ER membranes while enabling interactions with dislocation machinery. OS9 binds high-mannose oligosaccharides on terminally misfolded proteins through its MRH lectin domain, recruiting them to the Hrd1-SEL1L E3 ubiquitin ligase complex via direct SEL1L interaction, which delivers substrates to the dislocation channel for retrotranslocation into the cytosol followed by proteasomal degradation. Binding specificity arises from calcium coordination within the MRH domain that positions mannose residues for recognition, distinguishing misfolded conformers from properly folded glycoproteins during calnexin/calreticulin cycle reglucosylation events. OS9 forms a stable complex with XTP3-B to enhance recognition of nonglycosylated substrates through hydrophobic patch exposure, while IRE1/XBP1 pathway activation transcriptionally upregulates both OS9 isoforms during acute ER stress to amplify degradation capacity. The protein colocalizes with ERGIC-53 and VIP36 cargo receptors to prevent anterograde transport of terminally misfolded α1-antitrypsin variants and the null Hong Kong variant. OS9 deficiency impairs the degradation of destabilized CFTR folding mutants and ricin toxin A chain, leading to cytosolic accumulation. Isoform-specific expression patterns emerge through alternative splicing, with OS9.1 predominant in fibroblasts and OS9.2 enriched in professional secretory cells. Dysregulation of OS9 is linked to congenital disorders of glycosylation and protein folding diseases, including α1-antitrypsin deficiency.

Background

OS9 functions as an endoplasmic reticulum resident lectin within the mannose-6-phosphate receptor homology domain family, recognizing monoglucosylated N-glycans on misfolded glycoproteins to facilitate ER-associated degradation. The protein contains an N-terminal signal sequence, a central MRH domain with mannose-binding sites flanked by conserved charged residues, and C-terminal transmembrane and cytoplasmic tail regions that anchor it to ER membranes while enabling interactions with dislocation machinery. OS9 binds high-mannose oligosaccharides on terminally misfolded proteins through its MRH lectin domain, recruiting them to the Hrd1-SEL1L E3 ubiquitin ligase complex via direct SEL1L interaction, which delivers substrates to the dislocation channel for retrotranslocation into the cytosol followed by proteasomal degradation. Binding specificity arises from calcium coordination within the MRH domain that positions mannose residues for recognition, distinguishing misfolded conformers from properly folded glycoproteins during calnexin/calreticulin cycle reglucosylation events. OS9 forms a stable complex with XTP3-B to enhance recognition of nonglycosylated substrates through hydrophobic patch exposure, while IRE1/XBP1 pathway activation transcriptionally upregulates both OS9 isoforms during acute ER stress to amplify degradation capacity. The protein colocalizes with ERGIC-53 and VIP36 cargo receptors to prevent anterograde transport of terminally misfolded α1-antitrypsin variants and the null Hong Kong variant. OS9 deficiency impairs the degradation of destabilized CFTR folding mutants and ricin toxin A chain, leading to cytosolic accumulation. Isoform-specific expression patterns emerge through alternative splicing, with OS9.1 predominant in fibroblasts and OS9.2 enriched in professional secretory cells. Dysregulation of OS9 is linked to congenital disorders of glycosylation and protein folding diseases, including α1-antitrypsin deficiency.

References
https://pubmed.ncbi.nlm.nih.gov/18264092/

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

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

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%