The ELISA Detection of The Human Attractin Protein
The ELISA detection of the human Attractin protein is a core method in scientific research for quantifying its expression level and correlating with disease mechanisms. It involves two types of detection: secretory type (in plasma/body fluids) and membrane-bound type in cell lysates.
Detection classification
1. Secreted Attractin ELISA (Most commonly used, suitable for body fluid samples)
Adapted samples: Human plasma, serum, cerebrospinal fluid, cell culture supernatant, saliva and other soluble samples
Core advantages: Secreted proteins are easily soluble in body fluids and do not require complex processing. The ELISA detection sensitivity is high (can detect ng~pg levels), has good repeatability, and is suitable for screening large sample size clinical cohorts/animal experiment samples.
Key points to note: Samples should be stored at low temperature (4℃) for a short period and frozen at -80℃ for long-term storage. Avoid repeated freezing and thawing (≤3 times); For plasma samples, EDTA anticoagulant should be used to avoid protein degradation during the coagulation process; Before sample testing, dilute the samples proportionally with the diluent provided by the kit to eliminate the matrix effect.
2. Membrane-bound Attractin ELISA (for cell/tissue samples)
Adjuvant samples: Primary cells (such as oligodendrocytes, melanocytes), cell line lysates, tissue homogenate supernatants
Core premise: For membrane-bound type, cells/tissues need to be lysed first, and total protein or membrane protein components need to be extracted. It is necessary to ensure that the lysis buffer (commonly RIPA + protein inhibitors) has no protein hydrolytic activity. The entire process should be carried out at low temperature to prevent protein degradation; after extraction, the protein concentration needs to be determined (using BCA method) to ensure consistent total protein loading, reducing experimental errors.
Key points to note: Membrane proteins are hydrophobic, so an appropriate amount of detergent (such as Triton X-100) should be added to the lysis buffer, and it is necessary to confirm that the ELISA kit is compatible with the detergent during detection to avoid affecting the antigen-antibody binding.
Core application
1. Research on neurological disorders (such as leukodystrophy, multiple sclerosis, etc.)
Core application: Detect the content of Attractin protein in the cerebrospinal fluid and peripheral blood of patients, compare with the healthy control group, and verify the association between "down-regulation of protein expression and myelin damage"; in animal models (such as mice with myelin injury), dynamically detect the protein levels in cerebrospinal fluid/brain tissue homogenates at different disease progression stages, and clarify their correlation with disease progression.
Experimental value: ELISA quantitative data can serve as a basis for screening potential biomarkers for disease diagnosis, supporting the mechanism hypothesis of "Attractin deficiency leads to myelin abnormalities".
2. Metabolic Disease Research (Obesity, Type 2 Diabetes)
Core Application: Measure the serum Attractin levels in obese/diabetic patients, combined with genotyping (ATRN gene polymorphism), to analyze the correlation between "specific gene variations + protein expression levels" and the degree of obesity, insulin resistance index (HOMA-IR); In cell experiments, detect the changes in the release amount of secreted Attractin in adipocytes/liver cells stimulated by insulin.
Experimental Value: Clarify the correlation between protein expression levels and metabolic indicators, providing quantitative evidence for "Attractin regulating energy homeostasis".
3. Research on Immune Inflammatory Diseases (Rheumatoid Arthritis, Autoimmune Diseases)
Core Application: Measure the content of Attractin in patients' serum and joint cavity effusion, compare the protein differences between the inflammatory active period and the remission period, and verify the correlation between "high protein expression and inflammation amplification"; In vitro experiments, detect the expression changes of secreted Attractin in macrophages/lymphocytes stimulated by inflammatory factors (such as TNF-α, IL-6).
Experimental Value: Quantify the expression fluctuations of proteins under inflammatory conditions, support its function research as an inflammatory regulatory factor, and can also be used to screen the impact of anti-inflammatory drugs on its expression.
4. Pigment Abnormality Disease Research (such as vitiligo)
Core Application: Cultivate human melanocytes in vitro, detect the content of secreted Attractin in the cell supernatant, and the level of membrane-bound protein in the cell lysate, and compare the expression differences between normal melanocytes and melanocytes from patients with vitiligo; In animal models, detect the correlation between protein expression levels in skin tissue homogenates and melanin synthesis amounts.
Experimental Value: Clarify the direct association between abnormal protein expression and impaired melanocyte function.
5. Rare genetic diseases (syndromic obesity)
Core application: Detect the protein level of Attractin in the peripheral blood and skin fibroblast lysate of the patient, combined with the mutation sites of the ATRN gene, to analyze the association between "reduced/absent expression of mutant protein" and the multiple phenotypes of the disease (obesity, cognitive impairment).
Experimental value: Provide direct quantitative evidence for the gene-protein-phenotype association of rare diseases.
Detection classification
1. Secreted Attractin ELISA (Most commonly used, suitable for body fluid samples)
Adapted samples: Human plasma, serum, cerebrospinal fluid, cell culture supernatant, saliva and other soluble samples
Core advantages: Secreted proteins are easily soluble in body fluids and do not require complex processing. The ELISA detection sensitivity is high (can detect ng~pg levels), has good repeatability, and is suitable for screening large sample size clinical cohorts/animal experiment samples.
Key points to note: Samples should be stored at low temperature (4℃) for a short period and frozen at -80℃ for long-term storage. Avoid repeated freezing and thawing (≤3 times); For plasma samples, EDTA anticoagulant should be used to avoid protein degradation during the coagulation process; Before sample testing, dilute the samples proportionally with the diluent provided by the kit to eliminate the matrix effect.
2. Membrane-bound Attractin ELISA (for cell/tissue samples)
Adjuvant samples: Primary cells (such as oligodendrocytes, melanocytes), cell line lysates, tissue homogenate supernatants
Core premise: For membrane-bound type, cells/tissues need to be lysed first, and total protein or membrane protein components need to be extracted. It is necessary to ensure that the lysis buffer (commonly RIPA + protein inhibitors) has no protein hydrolytic activity. The entire process should be carried out at low temperature to prevent protein degradation; after extraction, the protein concentration needs to be determined (using BCA method) to ensure consistent total protein loading, reducing experimental errors.
Key points to note: Membrane proteins are hydrophobic, so an appropriate amount of detergent (such as Triton X-100) should be added to the lysis buffer, and it is necessary to confirm that the ELISA kit is compatible with the detergent during detection to avoid affecting the antigen-antibody binding.
Core application
1. Research on neurological disorders (such as leukodystrophy, multiple sclerosis, etc.)
Core application: Detect the content of Attractin protein in the cerebrospinal fluid and peripheral blood of patients, compare with the healthy control group, and verify the association between "down-regulation of protein expression and myelin damage"; in animal models (such as mice with myelin injury), dynamically detect the protein levels in cerebrospinal fluid/brain tissue homogenates at different disease progression stages, and clarify their correlation with disease progression.
Experimental value: ELISA quantitative data can serve as a basis for screening potential biomarkers for disease diagnosis, supporting the mechanism hypothesis of "Attractin deficiency leads to myelin abnormalities".
2. Metabolic Disease Research (Obesity, Type 2 Diabetes)
Core Application: Measure the serum Attractin levels in obese/diabetic patients, combined with genotyping (ATRN gene polymorphism), to analyze the correlation between "specific gene variations + protein expression levels" and the degree of obesity, insulin resistance index (HOMA-IR); In cell experiments, detect the changes in the release amount of secreted Attractin in adipocytes/liver cells stimulated by insulin.
Experimental Value: Clarify the correlation between protein expression levels and metabolic indicators, providing quantitative evidence for "Attractin regulating energy homeostasis".
3. Research on Immune Inflammatory Diseases (Rheumatoid Arthritis, Autoimmune Diseases)
Core Application: Measure the content of Attractin in patients' serum and joint cavity effusion, compare the protein differences between the inflammatory active period and the remission period, and verify the correlation between "high protein expression and inflammation amplification"; In vitro experiments, detect the expression changes of secreted Attractin in macrophages/lymphocytes stimulated by inflammatory factors (such as TNF-α, IL-6).
Experimental Value: Quantify the expression fluctuations of proteins under inflammatory conditions, support its function research as an inflammatory regulatory factor, and can also be used to screen the impact of anti-inflammatory drugs on its expression.
4. Pigment Abnormality Disease Research (such as vitiligo)
Core Application: Cultivate human melanocytes in vitro, detect the content of secreted Attractin in the cell supernatant, and the level of membrane-bound protein in the cell lysate, and compare the expression differences between normal melanocytes and melanocytes from patients with vitiligo; In animal models, detect the correlation between protein expression levels in skin tissue homogenates and melanin synthesis amounts.
Experimental Value: Clarify the direct association between abnormal protein expression and impaired melanocyte function.
5. Rare genetic diseases (syndromic obesity)
Core application: Detect the protein level of Attractin in the peripheral blood and skin fibroblast lysate of the patient, combined with the mutation sites of the ATRN gene, to analyze the association between "reduced/absent expression of mutant protein" and the multiple phenotypes of the disease (obesity, cognitive impairment).
Experimental value: Provide direct quantitative evidence for the gene-protein-phenotype association of rare diseases.
