recombinant human eif4e protein fused to his-tag at n-terminus  (Creative BioMart)

Journal: BMC Molecular and Cell Biology

Article Title: Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II

doi: 10.1186/s12860-020-00303-z

Figure Lengend Snippet: Presence of caps on 18S and 25S RNAs from stationary and BMH21 treated cells. a SYBR-gold stained gel showing that decapping did not affect RNA integrity. b Immunoblot of the gel ( a ) showing decrease in intensity after treatment with decapping enzyme. Some cross reactivity can be seen in lanes 1 and 3 (see text). c SYBR gold stained gel and corresponding Northern blot showing decapping of 18S, 25S and mRNAs. d SYBR gold stained gel showing total RNA extracted from mid-log (ML) and stationary (ST) C. albicans (lanes 2 and 3) and immunoblot (lanes 4 and 5) using anti-m7G-cap mAb to detect bands precipitated by cap binding protein (CBP) eIF4F. e Conditions in lanes 2 and 3 are the same as in ( d ), lanes 4 and 5 are untreated mid-log and stationary RNA. In lanes 6 and 7 RNA was decapped prior to precipitating with CBP, showing that decapping removes the target of eIF4E protein

Article Snippet: Two micrograms of total RNA from C. albicans were incubated with 1 μg of recombinant human eIF4E protein fused to His-tag at N-terminus (Creative BioMart) in binding buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT, 5 mM imidazole) and incubated at 4 °C overnight.

Techniques: Staining, Western Blot, Northern Blot, Binding Assay

Journal: BMC Molecular and Cell Biology

Article Title: Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II

doi: 10.1186/s12860-020-00303-z

Figure Lengend Snippet: CBP and RIP qPCR analysis. ( a and b ) RT-qPCR quantification of 18S, 25S and ITS-1 molecules precipitated from total RNA by ( a ) eIF4E (CBP) and (B) anti-m7G-cap mAb, both from mid-log (ML) and stationary (St) organisms. ITS-1 was used as a negative control for the assay. Error bars represent standard deviation from three different experiments. P values generated by Student’s test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Two micrograms of total RNA from C. albicans were incubated with 1 μg of recombinant human eIF4E protein fused to His-tag at N-terminus (Creative BioMart) in binding buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT, 5 mM imidazole) and incubated at 4 °C overnight.

Techniques: Quantitative RT-PCR, Negative Control, Standard Deviation, Generated