Hck Antibody (Rabbit mAb) [J18G8]

Application: Reactivity: Human

Lane 1: TF-1

Experiment Essentials

WB
Recommended WB dilution ratio: 1:20000

Usage Information

Dilution
WB1:20000

Application
WB

Reactivity
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
60 kDa

Positive Control	TF-1 cells; U-937 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 1:20000), 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 1:20000), 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
Hck Antibody (Rabbit mAb) [J18G8] detects endogenous levels of total Hck protein.

Subcellular Location
Cell junction, Cell membrane, Cytoskeleton, Golgi apparatus, Lysosome, Nucleus

Uniprot ID
P08631

Clone
J18G8

Synonym(s)
Tyrosine-protein kinase HCK, Hematopoietic cell kinase, Hemopoietic cell kinase, p59-HCK/p60-HCK, p59Hck, p61Hck, HCK

Background
Hck (hematopoietic cell kinase) is a myeloid-enriched Src family non-receptor tyrosine kinase that integrates signals from cytokine receptors, Fc receptors, integrins, and oncogenic tyrosine kinases to control proliferation, survival, polarization, and migration of myeloid and B-lineage cells. The protein shares the canonical Src family architecture, with an N-terminal myristoylated and palmitoylated membrane-targeting segment, followed by SH3 and SH2 domains that dock onto proline-rich motifs and phosphotyrosines on partner proteins, and a C-terminal kinase domain whose activity is constrained in the resting state by intramolecular engagement of a phosphorylated C-terminal tyrosine with the SH2 domain; dephosphorylation of this tail and engagement of activating receptors releases the autoinhibited conformation and permits substrate phosphorylation. In myeloid leukemias driven by BCR-ABL, Hck binds the fusion kinase via SH3/SH2 interactions, becomes catalytically activated, and directly phosphorylates STAT5 on its critical activation tyrosine, establishing a BCR-ABL–Hck–STAT5 axis that sustains expression of STAT5 target genes such as A1 and Pim-1 and promotes transformation and cytokine-independent growth of myeloid cells; pharmacologic inhibition or kinase-dead Hck abrogates this STAT5 activation and reduces leukemic cell viability. In acute myeloid leukemia carrying FLT3-ITD mutations, Hck operates downstream of mutant FLT3 and contributes to overexpression and activation of CDK6, linking receptor tyrosine kinase signaling through Hck to Cyclin D–CDK6–Rb control of G1 progression, and defining an essential FLT3–Hck–CDK6 pathway that underlies FLT3-ITD–dependent proliferation and sensitivity to CDK4/6 inhibitors. Hck also participates in the crosstalk between hematopoietic cells and the bone marrow niche through the CXCL12/CXCR4 axis, where its activity modulates adhesion, chemotaxis, and positioning of normal and leukemic progenitors within the marrow microenvironment, providing a mechanistic link between kinase signaling and niche-dependent leukemic cell retention and survival. Increased Hck expression and activity are detected in inflammatory joint and cartilage models, where Hck activation amplifies IL-1β–induced JAK–STAT3 signaling, upregulates IL-6 and TNF-α, promotes chondrocyte apoptosis, and accelerates extracellular matrix degradation, while Hck silencing dampens these responses and preserves tissue architecture, implicating this kinase as a driver of inflammatory tissue damage in osteoarthritis and as a candidate target in chronic joint disease. High HCK expression correlates with distinct transcriptional programs, immune cell infiltration signatures, poorer prognosis, and altered responses to antineoplastic agents, and selective Hck inhibition reduces PI3K/Akt and MAPK/ERK activation after erythropoietin or growth factor stimulation and preferentially induces death of dysplastic and leukemic progenitors while sparing normal hematopoietic stem cells.

Background

Hck (hematopoietic cell kinase) is a myeloid-enriched Src family non-receptor tyrosine kinase that integrates signals from cytokine receptors, Fc receptors, integrins, and oncogenic tyrosine kinases to control proliferation, survival, polarization, and migration of myeloid and B-lineage cells. The protein shares the canonical Src family architecture, with an N-terminal myristoylated and palmitoylated membrane-targeting segment, followed by SH3 and SH2 domains that dock onto proline-rich motifs and phosphotyrosines on partner proteins, and a C-terminal kinase domain whose activity is constrained in the resting state by intramolecular engagement of a phosphorylated C-terminal tyrosine with the SH2 domain; dephosphorylation of this tail and engagement of activating receptors releases the autoinhibited conformation and permits substrate phosphorylation. In myeloid leukemias driven by BCR-ABL, Hck binds the fusion kinase via SH3/SH2 interactions, becomes catalytically activated, and directly phosphorylates STAT5 on its critical activation tyrosine, establishing a BCR-ABL–Hck–STAT5 axis that sustains expression of STAT5 target genes such as A1 and Pim-1 and promotes transformation and cytokine-independent growth of myeloid cells; pharmacologic inhibition or kinase-dead Hck abrogates this STAT5 activation and reduces leukemic cell viability. In acute myeloid leukemia carrying FLT3-ITD mutations, Hck operates downstream of mutant FLT3 and contributes to overexpression and activation of CDK6, linking receptor tyrosine kinase signaling through Hck to Cyclin D–CDK6–Rb control of G1 progression, and defining an essential FLT3–Hck–CDK6 pathway that underlies FLT3-ITD–dependent proliferation and sensitivity to CDK4/6 inhibitors. Hck also participates in the crosstalk between hematopoietic cells and the bone marrow niche through the CXCL12/CXCR4 axis, where its activity modulates adhesion, chemotaxis, and positioning of normal and leukemic progenitors within the marrow microenvironment, providing a mechanistic link between kinase signaling and niche-dependent leukemic cell retention and survival. Increased Hck expression and activity are detected in inflammatory joint and cartilage models, where Hck activation amplifies IL-1β–induced JAK–STAT3 signaling, upregulates IL-6 and TNF-α, promotes chondrocyte apoptosis, and accelerates extracellular matrix degradation, while Hck silencing dampens these responses and preserves tissue architecture, implicating this kinase as a driver of inflammatory tissue damage in osteoarthritis and as a candidate target in chronic joint disease. High HCK expression correlates with distinct transcriptional programs, immune cell infiltration signatures, poorer prognosis, and altered responses to antineoplastic agents, and selective Hck inhibition reduces PI3K/Akt and MAPK/ERK activation after erythropoietin or growth factor stimulation and preferentially induces death of dysplastic and leukemic progenitors while sparing normal hematopoietic stem cells.

References
https://pubmed.ncbi.nlm.nih.gov/12411494/ https://pubmed.ncbi.nlm.nih.gov/26087188/

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%