The experiment was performed as previously described (42 (link)) with some modifications. The assay was performed in 40 mM HEPES-KOH, pH 7.5, 25 mM KCl, 8 mM MgCl2, 4 mM ATP, 1 mM glutamine, 10 nM GatCAB, and 32P-labeled Asp-tRNAAsn (various concentrations from 0.25 to 8.0 μM). The reaction mixture was kept on ice until the 2-min preequilibration step at 37°C and then initiated with ATP and glutamine. A 5-μl aliquot for each time point was removed and quenched with 5 μl nuclease P1 (Sigma; product no. N8630) in a 100-mM sodium citrate suspension (pH 4.7, 0.66 mg/ml). The digestion was kept in a 37°C heat block for 30 min, and 3 μl of sample was carefully spotted onto prewashed polyethyleneimine (PEI)-cellulose thin-layer chromatographic (TLC) plates (Merck Millipore; catalog no. 105725). The TLC plate was eluted in developing buffer containing 10 mM NH4Cl and 5% acetic acid for 100 to 140 min. The air-dried plates were exposed to phosphor screens for at least 16 h and then quantified by Typhoon FLA 9500 phosphorimager (Cytiva).
Typhoon fla 9500 phosphorimager
Manufactured by Cytiva
The Typhoon FLA 9500 phosphorimager is a versatile laboratory equipment designed for the detection and quantification of radiolabeled and fluorescent samples. It is capable of scanning a wide range of sample types, including gels, membranes, and microplates, to produce high-resolution digital images.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using typhoon fla 9500 phosphorimager
1
Quantitative Radioactive Assay for tRNA Aminoacylation
2
Asp-tRNA Asn Aminoacylation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiment was performed as previously described (23) with some modifications. The assay was performed in 40 mM HEPES-KOH pH 7.5, 25 mM KCl, 8 mM MgCl2, 4 mM ATP, 1 mM glutamine, 10 nM GatCAB and 32 P-labeled Asp-tRNA Asn (various concentration from 0.25-8.0 µM). The reaction was kept on ice until the 2 min pre-equilibration step at 37 °C, and then initiated with ATP and glutamine. A 5 µL aliquot for each time point was removed and quenched with 5 µL nuclease P1 (Sigma #N8630) in a 100 mM sodium citrate suspension (pH 4.7, 0.66 mg/mL). The digestion was kept in a 37 °C heat block for 30 min, and 3 µL of sample was carefully spotted onto pre-washed PEI-cellulose TLC plates (Merck Millipore, #105725). The TLC plate was eluted in developing buffer containing 10 mM NH4Cl and 5% acetic acid for 100-140 min. The air-dried plates were exposed to phosphor screens for at least 16 h, and then quantified by Typhoon FLA 9500 phosphorimager (Cytiva).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!