Coronin 1a/TACO Antibody (Rabbit mAb) [N18B9]

Application: Reactivity: Mouse, Rat, Human

Lane 1: Jurkat, Lane 2: Raji, Lane 3: MOLT4, Lane 4: Mouse brain

Usage Information

Dilution
WB1:1000 IHC1:1000 IF1:100 FCM1:500

Application
WB, IHC, IF, FCM

Reactivity
Mouse, Rat, Human

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
51 kDa 51 kDa,57 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	Human brain tissue; Rat spleen tissue; Human spleen tissue; Mouse brain tissue; Mouse spleen tissue; Human colon tissue; Jurkat cells; Raji cells; MOLT-4 cells; RAW 264.7 cells
Negative Control	HEK-293 cells

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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

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
Coronin 1a/TACO Antibody (Rabbit mAb) [N18B9] detects endogenous levels of total Coronin 1a/TACO protein.

Subcellular Location
Cytoplasm, Cytoplasmic vesicle, Cytoskeleton, Membrane

Uniprot ID
P31146

Clone
N18B9

Synonym(s)
CORO1, CORO1A, Coronin-1A, Coronin-like protein A, Coronin-like protein p57, Tryptophan aspartate-containing coat protein, Clipin-A, TACO

Background
Coronin 1A, also known as TACO, is a leukocyte-enriched actin-binding WD40-repeat protein of the coronin family that localizes to the cortical cytoskeleton and phagosomal membranes and functions as a regulator of actin dynamics, membrane trafficking and Ca²⁺-dependent signaling during immune responses and host–pathogen interactions. The protein contains an N‑terminal β‑propeller formed by WD repeats that binds F‑actin and Arp2/3, a more variable central region, and a C‑terminal coiled-coil segment implicated in oligomerization and interactions with signaling partners, allowing coronin 1A to bridge adjacent actin protomers and stabilize F‑actin while coordinating actin-remodeling factors at sites of membrane invagination and protrusion. Coronin 1A binds along actin filaments in a manner that can both stabilize filament bundles and modulate access of ADF/cofilin and Arp2/3, placing it as a hub that tunes leading-edge actin assembly, immunological synapse formation and motile behavior in hematopoietic cells. In T lymphocytes, coronin 1A links cytoskeleton plasticity to TCRαβ-induced signaling: loss of coronin 1A results in defective terminal development and survival of αβ T cells, excessive accumulation but reduced dynamics of F‑actin and WASP–Arp2/3 components at the immunological synapse, prolonged cell–cell contact, and impaired TCR signaling characterized by elevated basal JNK activation, reduced TCR-induced Ca²⁺ influx and defective IκB phosphorylation and degradation, indicating that coronin 1A is required to couple actin remodeling with proper engagement of Ca²⁺ and NF‑κB pathways. These defects translate into altered cytokine production and homeostasis of naïve T cells, and coronin 1A is a key factor in maintaining naïve T-cell survival and migration in response to chemokine cues, with recessive CORO1A mutations producing peripheral T-cell deficiency due to migration defects and increased apoptosis. In macrophages, coronin 1A/TACO plays a specialized role during mycobacterial infection: pathogenic mycobacteria recruit coronin 1A to the phagosomal membrane, where its continued presence prevents phagosome–lysosome fusion and allows bacilli to persist within nonacidified compartments. Coronin 1A is dispensable for general F‑actin-dependent processes such as phagocytosis, motility and membrane ruffling, but is required for activation of the Ca²⁺-dependent phosphatase calcineurin upon mycobacterial infection; in its absence, calcineurin activation fails, phagosomes proceed to lysosomal fusion and mycobacteria are killed, and pharmacologic inhibition of calcineurin similarly restores lysosomal delivery, defining a coronin 1A–calcineurin axis that specifically controls phagosome maturation in infected cells. Coronin 1A also modulates Rac1 signaling during cell migration by promoting a cytoskeletal-based feedback loop in which Rac1 activation drives actin remodeling that, in turn, maintains Rac1 at membranes and supports persistent motility, further integrating coronin 1A into Rho-family GTPase pathways that coordinate cell shape and movement. Coronin 1A also regulates lysosomal secretion in osteoclast-lineage cells, where it acts as a negative regulator of cathepsin K exocytosis and bone resorption by interfering with LC3 lipidation at the ruffled border and limiting lysosome–plasma membrane fusion in an actin-dependent fashion.

Background

Coronin 1A, also known as TACO, is a leukocyte-enriched actin-binding WD40-repeat protein of the coronin family that localizes to the cortical cytoskeleton and phagosomal membranes and functions as a regulator of actin dynamics, membrane trafficking and Ca²⁺-dependent signaling during immune responses and host–pathogen interactions. The protein contains an N‑terminal β‑propeller formed by WD repeats that binds F‑actin and Arp2/3, a more variable central region, and a C‑terminal coiled-coil segment implicated in oligomerization and interactions with signaling partners, allowing coronin 1A to bridge adjacent actin protomers and stabilize F‑actin while coordinating actin-remodeling factors at sites of membrane invagination and protrusion. Coronin 1A binds along actin filaments in a manner that can both stabilize filament bundles and modulate access of ADF/cofilin and Arp2/3, placing it as a hub that tunes leading-edge actin assembly, immunological synapse formation and motile behavior in hematopoietic cells. In T lymphocytes, coronin 1A links cytoskeleton plasticity to TCRαβ-induced signaling: loss of coronin 1A results in defective terminal development and survival of αβ T cells, excessive accumulation but reduced dynamics of F‑actin and WASP–Arp2/3 components at the immunological synapse, prolonged cell–cell contact, and impaired TCR signaling characterized by elevated basal JNK activation, reduced TCR-induced Ca²⁺ influx and defective IκB phosphorylation and degradation, indicating that coronin 1A is required to couple actin remodeling with proper engagement of Ca²⁺ and NF‑κB pathways. These defects translate into altered cytokine production and homeostasis of naïve T cells, and coronin 1A is a key factor in maintaining naïve T-cell survival and migration in response to chemokine cues, with recessive CORO1A mutations producing peripheral T-cell deficiency due to migration defects and increased apoptosis. In macrophages, coronin 1A/TACO plays a specialized role during mycobacterial infection: pathogenic mycobacteria recruit coronin 1A to the phagosomal membrane, where its continued presence prevents phagosome–lysosome fusion and allows bacilli to persist within nonacidified compartments. Coronin 1A is dispensable for general F‑actin-dependent processes such as phagocytosis, motility and membrane ruffling, but is required for activation of the Ca²⁺-dependent phosphatase calcineurin upon mycobacterial infection; in its absence, calcineurin activation fails, phagosomes proceed to lysosomal fusion and mycobacteria are killed, and pharmacologic inhibition of calcineurin similarly restores lysosomal delivery, defining a coronin 1A–calcineurin axis that specifically controls phagosome maturation in infected cells. Coronin 1A also modulates Rac1 signaling during cell migration by promoting a cytoskeletal-based feedback loop in which Rac1 activation drives actin remodeling that, in turn, maintains Rac1 at membranes and supports persistent motility, further integrating coronin 1A into Rho-family GTPase pathways that coordinate cell shape and movement. Coronin 1A also regulates lysosomal secretion in osteoclast-lineage cells, where it acts as a negative regulator of cathepsin K exocytosis and bone resorption by interfering with LC3 lipidation at the ruffled border and limiting lysosome–plasma membrane fusion in an actin-dependent fashion.

References
https://pubmed.ncbi.nlm.nih.gov/17632055/ https://pubmed.ncbi.nlm.nih.gov/18941544/

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%