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Anti-Tin2 Rabbit Antibody [K6G20]

Catalog No.: F3723

Application: Reactivity: Mouse, Rat, Human

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Mouse, Rat, Human

Source
Rabbit

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
50 kDa 38 kDa, 39 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight.

Positive Control	Rat thymus; Mouse thymus; Human thymus; Mouse brain; Mouse heart; Mouse kidney; Mouse spleen; NIH3T3; PC12; RAW264.7; C6; HUVEC; WI38; MCF7
Negative Control

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. 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/Nuclear 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/Nuclear 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 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.

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/Nuclear 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/Nuclear 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
Tin2 Rabbit mAb detects endogenous levels of total Tin2 protein.

Subcellular Location
Chromosome, Nucleus, Telomere

Uniprot ID
Q9BSI4

Clone
K6G20

Synonym(s)
TIN2, TINF2, TERF1-interacting nuclear factor 2, TRF1-interacting nuclear protein 2

Background
TIN2 (TRF1-interacting nuclear factor 2) is a central scaffold protein within the shelterin complex, a six-protein assembly that protects mammalian telomeres, regulating telomere length and integrity. TIN2 contains a telomere repeat factor homology (TRFH) domain, a TRFH-binding motif (TBM), and a domain associated with dyskeratosis congenita mutations. TIN2 interacts with TRF1 and TRF2, double-stranded telomeric DNA-binding proteins, through its TBM and TRFH domains, and directly binds TPP1 to facilitate the assembly of POT1 and TPP1 on telomeres, crucial for protecting single-stranded telomeric DNA. There are two major TIN2 isoforms, TIN2S and TIN2L, which modulate telomere stability and architecture. TIN2 coordinates cis (DNA compaction) and trans (DNA-DNA or DNA-RNA bridging) interactions, maintaining telomere structural integrity. Loss of TIN2 leads to telomere deprotection, activation of DNA damage responses (ATM/ATR kinases), chromatid fusion, and may result in embryonic lethality. TIN2 also interacts with the cohesin subunit SA1, regulating sister telomere cohesion, while Tankyrase1 controls TRF1-TIN2 association with telomeres. Acting as a cooperative platform, TIN2 bridges TRF1, TRF2, and TPP1, enabling shelterin functions in chromosome end protection, telomerase regulation, and genome stability. Mutations or dysfunction of TIN2 are linked to premature aging syndromes and cancer susceptibility.

Background

TIN2 (TRF1-interacting nuclear factor 2) is a central scaffold protein within the shelterin complex, a six-protein assembly that protects mammalian telomeres, regulating telomere length and integrity. TIN2 contains a telomere repeat factor homology (TRFH) domain, a TRFH-binding motif (TBM), and a domain associated with dyskeratosis congenita mutations. TIN2 interacts with TRF1 and TRF2, double-stranded telomeric DNA-binding proteins, through its TBM and TRFH domains, and directly binds TPP1 to facilitate the assembly of POT1 and TPP1 on telomeres, crucial for protecting single-stranded telomeric DNA. There are two major TIN2 isoforms, TIN2S and TIN2L, which modulate telomere stability and architecture. TIN2 coordinates cis (DNA compaction) and trans (DNA-DNA or DNA-RNA bridging) interactions, maintaining telomere structural integrity. Loss of TIN2 leads to telomere deprotection, activation of DNA damage responses (ATM/ATR kinases), chromatid fusion, and may result in embryonic lethality. TIN2 also interacts with the cohesin subunit SA1, regulating sister telomere cohesion, while Tankyrase1 controls TRF1-TIN2 association with telomeres. Acting as a cooperative platform, TIN2 bridges TRF1, TRF2, and TPP1, enabling shelterin functions in chromosome end protection, telomerase regulation, and genome stability. Mutations or dysfunction of TIN2 are linked to premature aging syndromes and cancer susceptibility.

References
https://pubmed.ncbi.nlm.nih.gov/39109533/ https://pubmed.ncbi.nlm.nih.gov/29160297/

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%