Kindlin-3 Antibody (Rabbit mAb) [B13M6]

Application: Reactivity: Human, Mouse, Rat

Lane 1: J774A.1

Usage Information

Dilution
WB1:1000 IP1:100 IHC1:400 FCM1:50

Application
WB, IP, IHC, 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
76 kDa N/A
^*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	Human non-Hodgkin's lymphoma tissue; Human colon carcinoma tissue; Human tonsil tissue; HEL9217 cells; CMK cells; RCH-ACV cells; J774A.1 cells; YB2/0 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.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
Kindlin-3 Antibody (Rabbit mAb) [B13M6] detects endogenous levels of total Kindlin-3 protein.

Subcellular Location
Cell junction, Cell projection

Uniprot ID
Q86UX7

Clone
B13M6

Synonym(s)
Fermitin family homolog 3; fermitin family homolog 3 (Drosophila); fermitin family member 3; FERMT3; KIND3; kindlin 3; Kindlin-3; MGC10966; MIG-2; Unc-112-related protein 2; UNC112C; URP2; URP2SF

Background
Kindlin-3 belongs to the kindlin family of FERM-domain focal adhesion adaptor proteins, and among the three mammalian paralogs its expression is restricted to hematopoietic lineages, distinguishing it from the more broadly expressed kindlin-1 and kindlin-2. Kindlin-3 binds the cytoplasmic tail of β-integrin subunits and acts together with talin-1 to drive integrin activation, but the two adaptors contribute distinct and separable steps within this process. Talin-1 engagement with the β2 integrin tail drives extension of the integrin ectodomain and opening of the headpiece, converting the receptor from a bent, low-affinity conformation to an extended one capable of ligand engagement. Kindlin-3 acts on a separate structural element, disrupting the membrane-proximal association between the integrin α and β cytoplasmic tails, a clasp that normally holds the receptor in its inactive state; release of this clasp permits unbending of the ectodomain and primes the receptor for high-affinity ligand binding. Ligand engagement by the extracellular domain, in the case of LFA-1 through interaction with ICAM-1, triggers transient association between the membrane-proximal region of the β2 tail and an N-terminal region of kindlin-3, and this ligand-induced disruption of the α/β clasp precedes and enables stable, high-affinity talin-1 binding to the β2 tail, a step that additionally requires the talin rod domain and an intact actomyosin cytoskeleton. This sequence establishes a positive feedback circuit in which kindlin-3 and talin-1 alternately reinforce integrin activation, coordinated by inside-out signaling through the GTPase Rap1 and by outside-in signaling generated once ligand-bound, high-affinity integrin conformers accumulate; mechanical shear force further stabilizes this active state and enhances the association of both adaptors with the receptor. In migrating lymphocytes, kindlin-3 and talin-1 distribute asymmetrically, tracking the spatial maturation of integrin affinity from the leading edge toward the adherent cell body and linking this activation cascade to directional migration and firm adhesion under flow. Loss-of-function mutations in the FERMT3 gene abolish this integrin-activating function in platelets and leukocytes, producing leukocyte adhesion deficiency type III, a disorder marked by defective platelet aggregation and impaired leukocyte arrest and transmigration at sites of inflammation, underscoring the non-redundant, combinatorial requirement for both adaptors in integrin-dependent adhesion.

Background

Kindlin-3 belongs to the kindlin family of FERM-domain focal adhesion adaptor proteins, and among the three mammalian paralogs its expression is restricted to hematopoietic lineages, distinguishing it from the more broadly expressed kindlin-1 and kindlin-2. Kindlin-3 binds the cytoplasmic tail of β-integrin subunits and acts together with talin-1 to drive integrin activation, but the two adaptors contribute distinct and separable steps within this process. Talin-1 engagement with the β2 integrin tail drives extension of the integrin ectodomain and opening of the headpiece, converting the receptor from a bent, low-affinity conformation to an extended one capable of ligand engagement. Kindlin-3 acts on a separate structural element, disrupting the membrane-proximal association between the integrin α and β cytoplasmic tails, a clasp that normally holds the receptor in its inactive state; release of this clasp permits unbending of the ectodomain and primes the receptor for high-affinity ligand binding. Ligand engagement by the extracellular domain, in the case of LFA-1 through interaction with ICAM-1, triggers transient association between the membrane-proximal region of the β2 tail and an N-terminal region of kindlin-3, and this ligand-induced disruption of the α/β clasp precedes and enables stable, high-affinity talin-1 binding to the β2 tail, a step that additionally requires the talin rod domain and an intact actomyosin cytoskeleton. This sequence establishes a positive feedback circuit in which kindlin-3 and talin-1 alternately reinforce integrin activation, coordinated by inside-out signaling through the GTPase Rap1 and by outside-in signaling generated once ligand-bound, high-affinity integrin conformers accumulate; mechanical shear force further stabilizes this active state and enhances the association of both adaptors with the receptor. In migrating lymphocytes, kindlin-3 and talin-1 distribute asymmetrically, tracking the spatial maturation of integrin affinity from the leading edge toward the adherent cell body and linking this activation cascade to directional migration and firm adhesion under flow. Loss-of-function mutations in the FERMT3 gene abolish this integrin-activating function in platelets and leukocytes, producing leukocyte adhesion deficiency type III, a disorder marked by defective platelet aggregation and impaired leukocyte arrest and transmigration at sites of inflammation, underscoring the non-redundant, combinatorial requirement for both adaptors in integrin-dependent adhesion.

References
https://pubmed.ncbi.nlm.nih.gov/34103420/ https://pubmed.ncbi.nlm.nih.gov/22431571/

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%