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OTUD5 Rabbit mAb

Catalog No.: F1344

Lane 1: K562
Lane 2: HCT116
Lane 3: A549
Lane 4: U2OS

Experiment Essentials

Subcellular Location: Nucleus.
WB
Recommending using RIPA/Nuclear Lysis Buffer to prepare lysates.

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Source
Rabbit

Reactivity
Human, Rat, Monkey, Bovine, Dog, Pig

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
75 kDa

Positive Control	293T; K-562; HCT 116; A549; U-2 OS; Jurkat; Daudi; RBL-2H3; C6
Negative Control	293T (transfected with siRNA targeting human OTUD5)

Expression & Treatment Conditions

Sample	Treatment Conditions
293T	Tansfection (siRNA targeting hμMan OTUD5)
COS-7	Low expression
Click to view more sample data

^*For predicted expression levels of this protein in various human-derived cells and tissues, please refer to: http://www.proteinatlas.org

Sample	Treatment Conditions

Exprimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/Nuclear 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.Add an appropriate volume of RIPA/Nuclear 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 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 862. 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 Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/Nuclear 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.Add an appropriate volume of RIPA/Nuclear 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 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

862. 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
OTUD5 Rabbit mAb recognizes endogenous levels of total OTUD5 protein.

Uniprot ID
Q96G74

Clone
F16F3

Background
OTUD5 (OTU Deubiquitinase 5) is a cysteine protease with deubiquitinase activity and a member of the ovarian tumor protease (OTU) family. The human OTUD5 protein consists of 571 amino acids with a molecular weight of approximately 60 kDa. It has two key structural domains: an OTU catalytic domain responsible for its deubiquitinase activity and a ubiquitin-interacting motif (UIM) domain, which is crucial for its function and interaction with proteins like STING, SPT16, TRIM25, TP53, and PDCD5. The N-terminal region (1-212 amino acids) of OTUD5 is essential for its interaction with UBR5 and YAP. OTUD5 is a key regulator of various physiological processes, including immune signaling, DNA damage repair, and cellular responses to stress. It negatively regulates type I interferon (IFN-I) expression by downregulating TRAF3 K63-linked polyubiquitination and promotes DNA repair by stabilizing Ku80 or regulating FACT-dependent transcription at damaged chromatin. In myocardial ischemia/reperfusion, OTUD5 is involved in preventing ferroptosis by protecting GPX4 from degradation. OTUD5 also exerts oncogenic effects by deubiquitinating proteins like TRIM25, TP53, PDCD5, and PTEN, influencing cancer progression through the mTOR and Hippo signaling pathways. Thus, OTUD5 is implicated in inflammation, cancer, genetic disorders, and cardiovascular diseases, making it a potential therapeutic target.

Background

OTUD5 (OTU Deubiquitinase 5) is a cysteine protease with deubiquitinase activity and a member of the ovarian tumor protease (OTU) family. The human OTUD5 protein consists of 571 amino acids with a molecular weight of approximately 60 kDa. It has two key structural domains: an OTU catalytic domain responsible for its deubiquitinase activity and a ubiquitin-interacting motif (UIM) domain, which is crucial for its function and interaction with proteins like STING, SPT16, TRIM25, TP53, and PDCD5. The N-terminal region (1-212 amino acids) of OTUD5 is essential for its interaction with UBR5 and YAP. OTUD5 is a key regulator of various physiological processes, including immune signaling, DNA damage repair, and cellular responses to stress. It negatively regulates type I interferon (IFN-I) expression by downregulating TRAF3 K63-linked polyubiquitination and promotes DNA repair by stabilizing Ku80 or regulating FACT-dependent transcription at damaged chromatin. In myocardial ischemia/reperfusion, OTUD5 is involved in preventing ferroptosis by protecting GPX4 from degradation. OTUD5 also exerts oncogenic effects by deubiquitinating proteins like TRIM25, TP53, PDCD5, and PTEN, influencing cancer progression through the mTOR and Hippo signaling pathways. Thus, OTUD5 is implicated in inflammation, cancer, genetic disorders, and cardiovascular diseases, making it a potential therapeutic target.

References
[1] Fu L, et al. Cells. 2023 Apr 14;12(8):1161. [2] Liu L, et al. Adv Sci (Weinh). 2023 Oct;10(28):e2301852.

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

* Indicates a Required Field

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%