Donkey Anti-Human IgG/Bio

**Western Blotting Protocol** 1. Prepare lysates in SDS-compatible lysis buffer containing protease inhibitors, clarify by centrifugation, and denature samples at 95–100°C for 5 minutes. 2. Resolve proteins by SDS-PAGE and transfer to PVDF or nitrocellulose membrane. 3. Block with 5% BSA or nonfat milk in TBST for 1 hour at room temperature. 4. Incubate the membrane with Donkey Anti-Human IgG/Bio diluted in antibody dilution buffer overnight at 4°C with gentle agitation. Recommended dilution: WB=1:1000-10000; IHC-P=1:100-500; IHC-F=1:100-1000; IF=1:100-1000; ELISA=1:1000-10000; FCM=1:100-1000. 5. Wash 3 times in TBST, then incubate with species-appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. 6. Wash again, develop with ECL substrate, and document target band intensity together with molecular weight marker and exposure conditions. **IHC-P Protocol** 1. Use formalin-fixed, paraffin-embedded sections mounted on charged slides. Bake, deparaffinize in xylene, and rehydrate through graded ethanol to water. 2. Perform heat-induced epitope retrieval in citrate or EDTA buffer, then cool and rinse in PBS or TBS. 3. Quench endogenous peroxidase if chromogenic detection will be used, and block nonspecific binding with serum or protein block for 20–30 minutes. 4. Incubate sections with Donkey Anti-Human IgG/Bio in antibody diluent for 1 hour at room temperature or overnight at 4°C. Recommended dilution: WB=1:1000-10000; IHC-P=1:100-500; IHC-F=1:100-1000; IF=1:100-1000; ELISA=1:1000-10000; FCM=1:100-1000. 5. Wash thoroughly, apply the appropriate secondary detection system, develop with DAB or equivalent substrate, counterstain with hematoxylin, dehydrate, mount, and compare specific staining with negative control tissue. **IHC-F Protocol** 1. Use fresh frozen sections fixed briefly with cold acetone or paraformaldehyde according to antigen sensitivity. 2. Bring slides to room temperature, wash in PBS, and block nonspecific binding before primary antibody incubation. 3. Apply Donkey Anti-Human IgG/Bio at the optimized dilution for 1 hour at room temperature or overnight at 4°C. Recommended dilution: WB=1:1000-10000; IHC-P=1:100-500; IHC-F=1:100-1000; IF=1:100-1000; ELISA=1:1000-10000; FCM=1:100-1000. 4. Wash in PBS or TBS, apply enzyme- or fluorophore-conjugated secondary antibody, and protect from light when fluorescence detection is used. 5. Mount with compatible medium and evaluate signal localization against isotype or no-primary control sections. **IF Protocol** Use assay conditions validated for IF, apply Donkey Anti-Human IgG/Bio at the recommended working dilution, include proper positive and negative controls, perform thorough wash steps, and document incubation time, temperature, and detection conditions for reproducible performance. Recommended dilution: WB=1:1000-10000; IHC-P=1:100-500; IHC-F=1:100-1000; IF=1:100-1000; ELISA=1:1000-10000; FCM=1:100-1000. **FCM Protocol** Use assay conditions validated for FCM, apply Donkey Anti-Human IgG/Bio at the recommended working dilution, include proper positive and negative controls, perform thorough wash steps, and document incubation time, temperature, and detection conditions for reproducible performance. Recommended dilution: WB=1:1000-10000; IHC-P=1:100-500; IHC-F=1:100-1000; IF=1:100-1000; ELISA=1:1000-10000; FCM=1:100-1000. **ELISA Protocol** 1. Coat the plate with antigen or capture reagent according to assay design, block with protein-containing buffer to reduce nonspecific binding, and incubate with Donkey Anti-Human IgG/Bio at the optimized working dilution. Recommended dilution: WB=1:1000-10000; IHC-P=1:100-500; IHC-F=1:100-1000; IF=1:100-1000; ELISA=1:1000-10000; FCM=1:100-1000. 2. Wash between each incubation using PBS or TBS containing a low concentration of Tween-20. 3. Add enzyme-conjugated secondary antibody, develop with appropriate substrate, stop the reaction at the recommended endpoint, and.