| Catalog # | G8599-04 |
| Applications | Suitable for use in ELISA and Immunoblot. Other applications not tested. |
| Recommended Dilution | Immunoblot Analysis: 2-4ug/ml detects Gia1 from a rat brain microsomal preparation. Rat brain microsomal preparation was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-Gia1 (2ug/ml). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP* and a chemiluminescence detection system. |
| Optimal dilutions to be determined by the researcher. |
| *Recommended secondary antibodies | I1904-06C: IgG, H&L (HRP) (X-Adsorbed) Pab Gt xMo |
| I1904-06H IgG, H&L (HRP) (X-Adsorbed) Pab Gt xMo |
| I1904-14C: IgG, Fc (HRP) Pab Gt xMo |
| I1904-19C: IgG, F(ab’)2 (HRP) Pab Gt xMo |
| Immunoblot Protocol | 1. Perform SDS-PAGE on a microsomal membrane preparation and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water. |
| 2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 60 minutes at RT with constant agitation. |
| 3. Incubate the nitrocellulose with 2-4ug/ml of anti-Gia1, G8599-04 diluted in freshly prepared PBS-MLK overnight with agitation at 4°C. |
| 4. Wash the nitrocellulose twice with water. |
| 5. Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-mouse HRP conjugated IgG, 1:3000 dilution was used)* in PBS-MLK for 1.5 hours at room temperature with agitation. |
| 6. Wash the nitrocellulose with water twice. |
| 7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 10 minutes. |
| 8. Rinse the nitrocellulose in 4-5 changes of water for 2 hours. |
| 9. Use detection method of choice (enhanced chemiluminescence was used). |
| Storage and Stability | May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
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| CAS Number | n/a |
| Clone Type | Monoclonal |
| Isotype | IgG2b |
| Clone No | 4i40 |
| Host | Mouse |
| Source | Human |
| Concentration | ~1mg/ml |
| Form | Supplied as a liquid in 0.07M Tris-glycine, pH 7.4, 0.105M NaCl, containing 30% glycerol. |
| Purity | Purified by Protein G affinity chromatography. |
| Immunogen | A synthetic peptide (CAGAAEEGFMTAELA) corresponding to amino acids 110-123 of Gia1 (KLH). |
| Specificity | Recognizes Gia1, Mr 41kD. Approximately 30% crossreactivity with Gia3. Species Crossreactivity: Broad species crossreactivity expected due to the highly conserved nature of Gia1. |
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| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
