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How to Cite 1. For In-Text Citation (Materials & Methods): 2. For Key Resources Table: |
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Biological Description
| Specificity | Carboxymethyl Lysine Antibody (Mouse mAb) [B15G14] detects endogenous levels of total Carboxymethyl Lysine modified protein. |
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| Background | Carboxymethyl lysine, also designated Nε-carboxymethyllysine or CML, is a non-enzymatic modification of the lysine side chain and represents the most extensively characterized advanced glycation end product, formed when reducing sugars react with free amino groups on proteins through the Maillard reaction. CML arises through three convergent chemical routes: oxidative cleavage of the Amadori rearrangement product formed early in the glycation reaction, fragmentation of the Schiff base intermediate through the Namiki pathway, and direct reaction of lysine residues with glyoxal generated during glucose autoxidation, with modeling of these competing pathways indicating that the glyoxal route dominates CML generation under most physiological glucose and oxidative conditions. Once formed, CML functions as a ligand for the receptor for advanced glycation end products, RAGE, a pattern-recognition receptor expressed on adipocytes, endothelial cells, and macrophages, and CML engagement of RAGE activates downstream signaling that converges on nuclear factor-κB, driving production of inflammatory mediators. In adipose tissue, RAGE-mediated accumulation of CML activates this CML-RAGE axis to dysregulate adipokine expression, and this mechanism links elevated CML directly to obesity-associated insulin resistance, positioning the CML-RAGE interaction as a specific molecular route connecting a glycation product to a defined metabolic disease phenotype rather than a generalized marker of tissue damage. Beyond adipose tissue, CML-RAGE engagement activates the ERK and NF-κB pathways to drive migration, invasion, and stemness in osteosarcoma cells, and CML accumulation correlates with advanced tumor stage, indicating that the same receptor-coupled signaling cascade operates across metabolic and oncological contexts depending on the tissue expressing RAGE. CML also accumulates in tissues under conditions of oxidative stress and elevated glucose, including diabetic hearts and fatty liver, where its endogenous formation induces inflammatory marker expression independent of dietary intake, while dietary CML formed during thermal food processing contributes an additional, exogenous source that adds to the endogenous pool. Because CML formation depends on reactive carbonyl intermediates including glyoxal, methylglyoxal, and glucosone rather than on a single fixed precursor, its generation is sensitive to oxidative conditions, transition metal availability, and thermal processing, making CML both a chemically well-defined marker of cumulative glycoxidative burden and a functionally active RAGE ligand relevant to researchers studying diabetic complications, obesity-associated inflammation, and RAGE-driven tumor signaling. |
Usage Information
| Application | WB, IHC, FCM, ELISA | Dilution |
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| Reactivity | All | ||||||||
| Source | Mouse Monoclonal Antibody | MW | |||||||
| 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 | ||||||
| WB |
Experimental Protocol:
Sample preparation
1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold 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 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 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. 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. Reference Table for Selecting PVDF Membrane Pore Size Specifications ( 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.
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| 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.
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References
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Application Data
WB
Validated by Selleck
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Lane 1: Hela, Lane 2: Hela (methylglyoxal, 2 mM, 24 h)