CD54/ICAM-1 Antibody (Rabbit mAb) [L16P13]

Application: Reactivity: Mouse

Lane 1: Mouse spleen, Lane 2: Mouse liver, Lane 3: Mouse kidney

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Usage Information

Dilution
WB1:1000 IHC1:2000

Application
WB, IHC

Reactivity
Mouse

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
58 kDa 80-110 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	Mouse liver tissue; Mouse kidney tissue; Mouse spleen tissue; Mouse lung tissue; bEnd.3 cells; A20 cells; RAW 264.7 cells; WEHI-231 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min. 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.

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
CD54/ICAM-1 Antibody (Rabbit mAb) [L16P13] detects endogenous levels of total CD54/ICAM-1 protein.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
P05362

Clone
L16P13

Synonym(s)
CD54 \| Intercellular adhesion molecule 1 \| ICAM-1 \| MALA-2 \| MyD10 \| Icam1

Background
ICAM1, or intercellular adhesion molecule 1, is an immunoglobulin superfamily cell-adhesion receptor expressed at low basal levels on endothelial cells and various leukocytes and strongly inducible by proinflammatory cytokines, where it serves as a central organizer of leukocyte trafficking and immune synapse formation. The ectodomain is composed of multiple Ig-like domains that present binding surfaces for β2 integrins, including ITGAL:ITGB2 (LFA‑1) and ITGAM:ITGB2 (Mac‑1), as well as other ligands such as fibrinogen and hyaluronan, while the short cytoplasmic tail connects to the actin cytoskeleton and signaling adaptors to support outside‑in signaling during adhesion. Engagement of ICAM1 by LFA‑1 on T cells stabilizes T cell–endothelial and T cell–antigen-presenting cell contacts and contributes to formation of the immunological synapse, providing a calibrated adhesion system that allows T‑cell receptor signaling strength to be translated into stable conjugate formation, costimulation and efficient cytotoxic or helper responses. Binding of Mac‑1 on activated neutrophils to ICAM1 on stimulated endothelium under shear stress supports firm adhesion and crawling, positioning neutrophils for trans-endothelial migration at inflamed sites. During leukocyte trans-endothelial migration, ICAM1 ligation triggers assembly of endothelial “apical cups” enriched in F‑actin and caveolae by recruiting ARHGEF26/SGEF and activating the small GTPase RHOG, which reshapes the endothelial surface around adherent leukocytes and facilitates diapedesis. ICAM1 engagement also activates endothelial nitric oxide synthase through Ca²⁺ and AMP‑activated protein kinase, linking adhesion to vasoregulatory and barrier-signaling programs that further influence leukocyte passage. At the signaling level, ICAM1 crosslinking induces tyrosine phosphorylation cascades, kinase activation and transcription factor engagement, leading to changes in cytokine production, membrane protein expression, reactive oxygen species generation and, in some contexts, proliferation, making it more than a passive adhesion anchor. Transcriptional regulation of ICAM1 expression is driven by promoter elements including a κB site responsive to TNF, IL‑1, LPS and phorbol esters, and expression is inhibited by glucocorticoids, placing ICAM1 within NF‑κB–dependent inflammatory gene programs that can be pharmacologically modulated. ICAM1 is robustly expressed on epithelial and tumor cells, where it participates in cell–cell aggregation, for example through interaction with the mucin MUC1, and contributes to antigen presentation and cytotoxic T‑cell activation via costimulatory interactions with T‑cell integrins. Shed soluble ICAM1 is detectable in plasma and is elevated in many pathological states including malignancies, autoimmune and allergic diseases, atherosclerosis, ischemia, and transplant rejection, making ICAM1 a useful biomarker for inflammatory and immune dysregulation.

Background

ICAM1, or intercellular adhesion molecule 1, is an immunoglobulin superfamily cell-adhesion receptor expressed at low basal levels on endothelial cells and various leukocytes and strongly inducible by proinflammatory cytokines, where it serves as a central organizer of leukocyte trafficking and immune synapse formation. The ectodomain is composed of multiple Ig-like domains that present binding surfaces for β2 integrins, including ITGAL:ITGB2 (LFA‑1) and ITGAM:ITGB2 (Mac‑1), as well as other ligands such as fibrinogen and hyaluronan, while the short cytoplasmic tail connects to the actin cytoskeleton and signaling adaptors to support outside‑in signaling during adhesion. Engagement of ICAM1 by LFA‑1 on T cells stabilizes T cell–endothelial and T cell–antigen-presenting cell contacts and contributes to formation of the immunological synapse, providing a calibrated adhesion system that allows T‑cell receptor signaling strength to be translated into stable conjugate formation, costimulation and efficient cytotoxic or helper responses. Binding of Mac‑1 on activated neutrophils to ICAM1 on stimulated endothelium under shear stress supports firm adhesion and crawling, positioning neutrophils for trans-endothelial migration at inflamed sites. During leukocyte trans-endothelial migration, ICAM1 ligation triggers assembly of endothelial “apical cups” enriched in F‑actin and caveolae by recruiting ARHGEF26/SGEF and activating the small GTPase RHOG, which reshapes the endothelial surface around adherent leukocytes and facilitates diapedesis. ICAM1 engagement also activates endothelial nitric oxide synthase through Ca²⁺ and AMP‑activated protein kinase, linking adhesion to vasoregulatory and barrier-signaling programs that further influence leukocyte passage. At the signaling level, ICAM1 crosslinking induces tyrosine phosphorylation cascades, kinase activation and transcription factor engagement, leading to changes in cytokine production, membrane protein expression, reactive oxygen species generation and, in some contexts, proliferation, making it more than a passive adhesion anchor. Transcriptional regulation of ICAM1 expression is driven by promoter elements including a κB site responsive to TNF, IL‑1, LPS and phorbol esters, and expression is inhibited by glucocorticoids, placing ICAM1 within NF‑κB–dependent inflammatory gene programs that can be pharmacologically modulated. ICAM1 is robustly expressed on epithelial and tumor cells, where it participates in cell–cell aggregation, for example through interaction with the mucin MUC1, and contributes to antigen presentation and cytotoxic T‑cell activation via costimulatory interactions with T‑cell integrins. Shed soluble ICAM1 is detectable in plasma and is elevated in many pathological states including malignancies, autoimmune and allergic diseases, atherosclerosis, ischemia, and transplant rejection, making ICAM1 a useful biomarker for inflammatory and immune dysregulation.

References
https://pubmed.ncbi.nlm.nih.gov/8834767/ https://pubmed.ncbi.nlm.nih.gov/10924857/

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%