![]() ![]() HRP-conjugated goat anti mouse IgG (heavy and light chain) secondary antibody ( STAR207P) was used at 1/10,000 in lanes 2 and 3. Lanes 3 and 6 were used as controls to detect the background signaling of the secondary detection reagent (no primary antibody control). Mouse anti actin gamma ( MCA5776GA) was used to detect actin gamma (approximate MW of 46 kDa) in lanes 2 and 5 only. Bio-Rad Precision Plus Protein Prestained Standards were run in lanes 1 and 4. Mouse thymus lysate (25 μg) was loaded onto a 4–15% Criterion™ TGX Stain-Free™ Protein Gel (lanes 2, 3, 5, and 6) and proteins were transferred onto a nitrocellulose membrane. Arrow points to GAPDH (molecular weight 37 kDa). HRP conjugated Tidyblot ( STAR209P) was used at 1/200 and visualized on the Bio-Rad Chemidoc Touch Imaging System. Rabbit anti GAPDH antibody ( VPA00187) was used at 1/1000 in lanes 1-5. 8.75 µl Jurkat whole cell lysate (WCL) was run in lanes 5 & 11 while Precision Plus Protein™ Prestained Standards were run in lane 6. IP was performed on Jurkat cell lysates using 10 μg (lanes 1 & 7), 5 μg (lanes 2 & 8), 1 μg (lanes 3 & 9) Mouse anti GAPDH antibody ( MCA4739), and 10 μg Mouse IgG1 Negative Control (lanes 4 & 10) ( MCA1209).Ĩ.75 μl of each IP was loaded onto an AnykD Criterion TGX Stain-Free gel. Western blot analysis of GAPDH IP samples. Visualization was carried out using the Bio-Rad ChemiDoc with automatic exposure. ![]() TidyBlot was used at 1/200 as the secondary detection reagent. Rabbit anti Human GAPDH ( AHP1628) was used as the primary antibody at a dilution of 1/1000 in lane 2. Bio-Rad Precision Plus molecular weight marker was run in lane 1. HeLa (lane 2) cell lysate was run under reducing conditions on SDS PAGE using the Bio-Rad V3 system and transferred to a PVDF membrane. The TidyBlot Western Blot Detection Reagent:HRP only bound to the native actin gamma antibody whereas the standard anti-Mouse IgG heavy and light recognized both the native and denatured antibodies The Blot was visualized with the Bio-Rad Chemidoc Touch Imaging System. TidyBlot Western Blot Detection Reagent:HRP was used as the secondary reagent in lanes 2 and 3 and an HRP conjugated anti-mouse IgG heavy and light chain antibody was used as the secondary in lanes 5 and 6. Lanes 3 and 6 were used as controls to detect the background signaling of the secondary detection agent (no primary antibody control). Mouse anti actin gamma ( VMA00049) was used to detect actin gamma (approximate MW of 46 kDa) in lanes 2 and 5 only. ![]() Bio-Rad Precision Plus MW marker was run in lanes 1 and 4. 25 μg of HeLa cell lysate spiked with 3.5 μg of mouse IgG was run in lanes 2, 3, 5 and 6 under reducing conditions on SDS PAGE using the Bio-Rad V3 system and transferred to PVDF membrane. ![]()
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