hi, as a comment to zhijie's question, the biggest problem with marion bradford's "protein" assay is that it measures the SDS concentration too! therefore, where it would be most useful – that is in quantifying the total protein in an SDS-treated gel sample – it cannot be used! the amido-black method is far better, at least as far as i know. does anyone know why it's seldom used? our protocol is below if anyone's interested. best jon
Amido-Black assay. Dilute 10µl of the clarified protein extract (also in SDS sample buffer) to 200µl with water, add 600µl Amido-Black reagent, mix and incubate at RT for 5min. Centrifuge for 5min at 13krcf. Discard the supernatant and wash the pellet twice in 500µl Acid-MeOH [10%(v/v) HAc in MeOH]. The final supernatant should be colourless. Dissolve the pellet in 1ml 1M NaOH (warm if necessary) and assay at 615nm. Amido-Black reagent Stock = 0.13g Amido-Black 10B (Merck) + 1ml HAc + 9ml MeOH. Stir well then filter. Stable at 4°C indefinitely. To use, dilute 1ml of this stock in 50ml Acid-MeOH. Stable upto 1 week at 4°C. Calibrate with a dilution series of a "standard" protein such as BSA. Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Alex Lee Gesendet: Freitag, 5. Oktober 2018 05:37 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] [Offtopic] Protein stain in Coomassie Blue dyes Hi All, Thanks for all of your inputs! Alex On Thu, Oct 4, 2018 at 7:31 PM Zhijie Li <zhijie...@utoronto.ca<mailto:zhijie...@utoronto.ca>> wrote: Hi, At high concentration (1-2%) the published saturating SDS:protein binding ratio is about 1.4:1 by weight, that is roughly one SDS molecule per two aa on average. It is dense but not that dense to prevent any further interaction. More importantly, as a quite hydrophilic small molecule SDS should have no trouble dissociating from the peptide when its in-solution concentration drops (therefore you can use SDS gel bands for MS). With common procedure, during staining the SDS should partially fall off( especially if the gel is heated), and partially remain with the protein in the gel, depending on: how hydrophobic the protein is, how low the environmental SDS concentration becomes, how much organic solvent there is in the solution, etc.. The coomassie should simply find whatever hydrophobic/positively charged patch to bind and aggregate. Besides, coomassie-R is probably slightly more hydrophobic than SDS so it is capable of competing SDS off if necessary. (The even less hydrophobic Coomassie G250 definitely binds protein in the presence of detergents - that how Blue Native gel works for membrane proteins) Finally, since the only thing you are looking for is some deeper blue to indicate the presence of protein, even if SDS did prevent dye binding to some extent, your gel still will work. This is different from when you want to use the dye to do some quantitative work such as the Bradford assay. It would be interesting to know the effect of detergents on Bradford. Zhijie On Oct 4, 2018, at 9:26 PM, Alex Lee <alexlee198...@gmail.com<mailto:alexlee198...@gmail.com>> wrote: Dear All, I am thinking that in an SDS-PAGE experiment, if protein samples are boiled in SDS containing loading dye, and supposedly SDS interacts with proteins, why the Coomassie Blue dyes could still interact with and stain the proteins? I am thinking SDS is covering the proteins, making no room for the Coomassie Blue dyes interaction. I'd appreciate it if any input from this forum. Alex ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
