get_conc_A280.Rd
Get protein's concentration from a dilution series measured with an
A200-1000 absorbance spectrum. ... Function expects an input such as the
csv file exported from plot_absorbance_spectrum
called '_processed.csv',
which contains values corrected for path length and normalised to blanks as a
column called normalised_cm1_value
, but retains replicate data containing
positional (well) information required for exporting predicted concentrations
at the end of this function. ... Function uses
fpcountR::get_extcoeff_A280
to get EC in M-1cm-1 and wavelength,
and converts it to an EC mass extinction coefficient in (mgml)-1cm-1 using
the MW (worked out from protein_seq
and fpcountR::get_mw
). Then
the function uses the EC_A280_mgml to work out the concentration of protein
in each well, using three correction methods. Instead of using the normalised
data directly, the values used are based on a LOESS fit through the
absorption spectra to minimise fluctuations due to noise. ... Finally, linear
models are fitted to each concentration prediction method, and a csv file is
built containing predicted concentrations according to the user's chosen
correction method. Plots showing each of the analytical steps are saved
concurrently.
get_conc_A280( protein_slug, protein_seq, buffer = "", processed_spectrum_csv, wells_to_remove = NULL, disulphides = FALSE, showWarnings = TRUE, showMessages = FALSE, corr_method = "none", wav_to_use1 = 340, wav_to_use2 = 333, outfolder )
protein_slug | character string of protein name in 'slug' form to match slug of FPbase entry. |
---|---|
protein_seq | character string of protein sequence using 1-letter code. Required for MW calculation. |
buffer | character string of buffer. Optional. Defaults to "". |
processed_spectrum_csv | Path to csv file of a processed absorbance
spectrum. Processing should be done with |
wells_to_remove | list of wells to remove before analysis. Defaults to NULL. |
disulphides | required for calculation of A280 extinction coefficient. logical. Does protein have disulphides? Defaults to FALSE. |
showWarnings | required for calculation of A280 extinction coefficient. logical. Should function show warnings? Defaults to TRUE. |
showMessages | required for calculation of A280 extinction coefficient. logical. Should function show messages? Defaults to TRUE. |
corr_method | string corresponding to type of correction method to use
for the data to remove contribution of light scatter. Options are |
wav_to_use1 | numerical value of wavelength (nm) to use for |
wav_to_use2 | numerical value of wavelength (nm) to use for |
outfolder | path to folder where output files should be saved. Defaults to current working directory. |
a280_concs <- get_conc_A280(protein_slug = "mcherry", protein_seq = protein_seq, buffer = "T5N15_pi", processed_spectrum_csv = "abs_parsed_processed.csv", corr_method = "scatter", wav_to_use1 = 340, wav_to_use2 = 315, outfolder = "protquant_a280/mCherry_T5N15pi")