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As a control for antibody, cells where CXCR4 expression was suppressed using siRNA54 was also used.
The AIS simulates the generation of the antigens, the combination of the antigens and antibodies, and the selection, cloning and hypermutation of B cells, the maintenance of diverse antibody cells, the generation of the memory cells with high affinities and the death of the antibody cells with low affinities.
Basic research into how to isolate and clone single antibody cells has allowed us to discover that 10-12% of HIV-infected persons make broadly neutralizing antibodies.
In 2004, Epel and her team found that that psychological stress is significantly correlated with shorter telomere length in leukocytes, antibody cells that fight disease.
Following labelling with primary antibody, cells were washed and incubated with goat anti-mouse or anti-rabbit immunoglobulins conjugated with either Alexa Fluor® 488 or 594 (Invitrogen).
To visualize endogenous TRAF4 with 2H1 antibody, cells were fixed in 50% Methanol/50% Acetone for 15 minutes at −20°C; no permeabilization was needed in this case.
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To investigate acetylation and ubiquitination, FoxO1 antibody (Cell signaling) was used to precipitate FoxO1.
The slides were incubated overnight at 4 °C with the following primary antibodies (diluted at 1/1000): anti-14-3-3ζ anti-14-3-3ζ anti-14-3-3ζ anti-14-3-3ζ
Treated cell lysate was incubated with 10 μg FoxO1 antibody (Cell Signaling) (for FoxO1 Co-IP), 8 μg O-GlcNAc antibody (Cell Signaling) (for O-GlcNAc Co-IP), respectively on a rotator at 4 °C overnight.
FoxO1 antibody (cell signaling), O-GlcNAc antibody (cell signaling) and Protein A beads (nProtein A SepharoseTM 4 Fast flow, GE Healthcare Europe GmbH, Freiburg, Germany) were employed to precipitate the O-GlcNAcylated FoxO1.
Next, the blots were washed and incubated with Anti-Rabbit IgG, HRP-linked Antibody (Cell Signaling) or Anti-mouse IgG, HRP-linked Antibody (Cell Signaling) and detected with ECL Western Blotting Substrate (Pierce).
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