All other buffers, purification reagents, and chemicals used in this work were purchased from Sigma/Aldrich, unless otherwise stated.
Final primer extension was performed at 72°C for 10 min. PCR products were analyzed by 2.0% of agarose gel electrophoresis with TAE buffer before purification (Qiagen Corp., Germany).
Samples were diluted directly in EIA Buffer without purification.
Gal-7 and R74S were dialyzed in the same buffer after purification.
The buffer throughout purification and subsequent procedures was 0.1 M Tris-HCl, pH 7.6, with 5 mM EDTA.
Because the electron density was not consistent with any of the crystallization components, protein storage buffer, or purification buffers, it is likely that the formate ion was present in the heme used in the reconstitution of the apoprotein.
The samples in each group with different barcode adapters were gathered to a tube which had been added the wash buffer of purification kit beforehand.
The two differentially labeled reactions to be compared were mixed and buffer exchange, purification, and concentration was accomplished by microcon-10 (Amicon) filtration.
Briefly, the non-encapsulated GEM content in the supernatant before pore sealing (m1) and GEM content in the collected washing buffer during purification after pore sealing (m2) were determined by OD at 265 nm using a microplate reader (M5e, Molecular Device, USA) or HPLC (C18 column equipped with a K-2600 spectrophotometer).
DOI: http://dx.doi.org/10.7554/eLife.04463.006 We suspect that experimental factors such as the buffer conditions, purification methodology (i.e., presence or absence of affinity tags), and the method used to determine the protein concentration (Bradford/BCA or A280) might contribute to the variability in the ITC data between the two papers.
