Scheda tecnica

Descrizione biologica

Specificità	Melanoma Associated Antigen 100+ / 7 kDa Antibody [G24P23] rileva i livelli endogeni della proteina totale Melanoma Associated Antigen 100+ / 7 kDa.
Contesto	Le proteine Melanoma Associated Antigen 100+ / 7 kDa (MAGE) sono antigeni associati ai tumori, tipicamente ristretti alle cellule germinali del testicolo nei tessuti adulti normali ma espresse in modo aberrante in vari tumori, tra cui melanoma, glioma, polmone, vescica e mieloma multiplo. La designazione “100+/7 kDa” si riferisce a varianti o isoforme con pesi molecolari intorno a 100 kDa e 7 kDa. Strutturalmente, le proteine MAGE condividono un dominio di omologia MAGE (MHD) conservato di circa 200 amminoacidi, composto da due domini a elica alata che formano una profonda fessura per il legame di peptidi o proteine, fiancheggiati da regioni N- e C-terminali variabili. Funzionalmente, le proteine MAGE possono agire come driver oncogenici interagendo con RING-type E3 ubiquitin ligases, modulando le vie di degradazione proteica che regolano apoptosi, proliferazione e metabolismo; ad esempio, MAGE-A3 promuove la sopravvivenza tumorale stimolando la degradazione di p53 mediata da TRIM28, mentre MAGE-A4 può indurre apoptosi tramite diversi partner proteici. La loro espressione tumore-specifica, plasticità strutturale e ruoli oncogenici rendono le MAGE sia biomarcatori che potenziali bersagli terapeutici nell'immunoterapia del cancro.

Specificità

Melanoma Associated Antigen 100+ / 7 kDa Antibody [G24P23] rileva i livelli endogeni della proteina totale Melanoma Associated Antigen 100+ / 7 kDa.

Contesto

Le proteine Melanoma Associated Antigen 100+ / 7 kDa (MAGE) sono antigeni associati ai tumori, tipicamente ristretti alle cellule germinali del testicolo nei tessuti adulti normali ma espresse in modo aberrante in vari tumori, tra cui melanoma, glioma, polmone, vescica e mieloma multiplo. La designazione “100+/7 kDa” si riferisce a varianti o isoforme con pesi molecolari intorno a 100 kDa e 7 kDa. Strutturalmente, le proteine MAGE condividono un dominio di omologia MAGE (MHD) conservato di circa 200 amminoacidi, composto da due domini a elica alata che formano una profonda fessura per il legame di peptidi o proteine, fiancheggiati da regioni N- e C-terminali variabili. Funzionalmente, le proteine MAGE possono agire come driver oncogenici interagendo con RING-type E3 ubiquitin ligases, modulando le vie di degradazione proteica che regolano apoptosi, proliferazione e metabolismo; ad esempio, MAGE-A3 promuove la sopravvivenza tumorale stimolando la degradazione di p53 mediata da TRIM28, mentre MAGE-A4 può indurre apoptosi tramite diversi partner proteici. La loro espressione tumore-specifica, plasticità strutturale e ruoli oncogenici rendono le MAGE sia biomarcatori che potenziali bersagli terapeutici nell'immunoterapia del cancro.

Informazioni sullutilizzo

Applicazione

IHC, FCM

Diluizione

IHC

1:5 - 1:20

Reattività

Human

Fonte

Mouse Monoclonal Antibody

MW

Tampone di conservazione

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Conservazione
(Dalla data di ricevimento)

-20°C (avoid freeze-thaw cycles), 2 years

Metodi sperimentali
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.

Metodi sperimentali

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.

Riferimenti

https://pubmed.ncbi.nlm.nih.gov/26910052/
https://pubmed.ncbi.nlm.nih.gov/23946777/

Dati di applicazione

IHC

Validato da Selleck

Immunohistochemical analysis of formalin fixed paraffin embedded human melanoma tissue with F3175 at 1:20 dilution.