Recombinant Anti-Ubiquitin (linkage-specific K27) antibody [EPR17034] (ab181537)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:2000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:2000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Ub conjugates from unstimulated or L18-MDP treated BMDMs were isolated by TUBE as described under purification of endogenous ub conjugates. After washing, the beads were re-suspended in 1× DUB dilution buffer. For Ubiquitin restriction analysis, Ub conjugates incubated without or with DUBs (USP2 [0.5 µM], OTUD3 [3 µM], Cezanne [2 µM], UCHL3 [3 µM], Trabid [3 µM], OTUB1 [10 µM], AMSH [3 µM], OTULIN [1 µM] and MYSM1 [2 µM]). All samples were incubated at 37 °C for 1 h with shaking and reaction was stopped by adding 2× Laemmli buffer. Samples were then resolved in 4–12% Nupage gradient gel (Life technology), followed by western blot analysis with anti- RIP2, and specific antibodieas against Ubiquitin linkages (M1, K27, K48, and K63). Representative uncropped ubiquitin blots for Fig. 1 and Fig. 5 are provided in Supplementary Figure 8 and Supplementary Figure 9 respectively.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Ubiquitin (linkage-specific K27) antibody [EPR17034] (ab181537) below.

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