Expasy ProtParam: pI (isoelectric point), extinction coefficient for UV-based concentration, etc.
ExPasy ProtParam is the place to go if you want to know the nitty-gritty about a protein you’re studying. It lets you calculate pI, molecular weight, extinction coefficient, & more). And you can access “wild-type” (normal version of a protein) info or you can paste in your own sequence, which is super helpful if you’re working with recombinant protein expression and have modified the protein to add an affinity tag for purification or removed a floppy part or something. So it’s a protein biochemist’s friend! Here’s a link to it: https://web.expasy.org/protparam/ It tells you a bunch of info. “Basic” things like how long the protein is (# of amino acids) and how “big” it is (molecular weight, in Daltons (Da). It even tells you the number of atoms! It also tells you about how “basic” the protein is - in terms of how many “basic” (i.e. usually positively-charged) amino acids a protein has. This will impact the pI (isoelectric point) which is the pH at which the protein is neutral overall. Go below that pH and there are “excess” protons available, and the protein will be positively-charged on average. Go about that pH and there are “too few” protons available, so the protein will be negatively-charged. ProtParam tells you the theoretical pI which is super useful for doing charge-based protein purification (i.e. ion exchange chromatography). If your protein has a low pI, we say it’s “acidic” and usually use anion exchange. If your protein has a high pI, we say it’s “basic” and usually use cation exchange. more on all this here: https://bit.ly/isoelectricpoint & https://youtu.be/CLgzYBm_ymk and more ion exchange chromatography here: blog form: http://bit.ly/ionexchangechromatography ; YouTube: https://youtu.be/RGF1l572IZY It also gives you the extinction coefficient. WAY more on this here: http://bit.ly/bradforduv & http://bit.ly/proteinmeasuring but the key thing is it allows you to calculate a (pure) protein’s concentration based on how much UV light it absorbs. Extinction coefficients tell you the absorbance value (A) that corresponds to 10 mg/mL (1%) or 1 mg/mL (0.1%). Those percentages come from the weight/volume percentage convention that a 1% solution corresponds to 1 g/100 mL - more on why here: http://bit.ly/weightvolume & https://youtu.be/uo0Lx_OmKBA You can calculate the estimated extinction coefficient using free online software tools like Expasy ProtParam. I say estimated because context matters - the local environment around the absorbing part can influence how eager it is to absorb a photon When I do this for BSA I see this: Extinction coefficients: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 42925 Abs 0.1% (=1 g/l) 0.638, assuming all pairs of Cys residues form cystines Ext. coefficient 40800 Abs 0.1% (=1 g/l) 0.607, assuming all Cys residues are reduced First it tells me the values under oxidizing conditions and below that it tells me the values under reducing conditions (the intracellular environment is reducing and we usually add reducing agents like DTT or β-mercaptoenthanol) to protein solutions to keep them happy outside the cell). It tells me this because cysteine crosslinks can also absorb, where applicable. Then I can plug this into Beer’s law (or have the computer do it for me) if I measure the absorbance. You measure this absorbance using something called a spectrophotometer. Basically it shines light through a solution and measures to what extent different wavelengths make it through (are transmitted) versus don’t make it through (are absorbed). This can be converted into concentration of solute (dissolved molecules) using Beer’s Law The equation is: A = εcl A = absorbance ε = extinction coefficient (aka molar absorptivity coefficient) - specific for particular molecule & particular wavelength; units of L mol-1cm-1 c = concentration (in mol/L) - this is molarity - a mole is just a chemist’s “baker’s dozen” - it’s Avogadro’s number (6.022 x 10^23) of something - solute molecules or donuts, it’s just a number http://bit.ly/solutionconcentrations l = path length (in cm) finished in comments
ExPasy ProtParam is the place to go if you want to know the nitty-gritty about a protein you’re studying. It lets you calculate pI, molecular weight, extinction coefficient, & more). And you can access “wild-type” (normal version of a protein) info or you can paste in your own sequence, which is super helpful if you’re working with recombinant protein expression and have modified the protein to add an affinity tag for purification or removed a floppy part or something. So it’s a protein biochemist’s friend! Here’s a link to it: https://web.expasy.org/protparam/ It tells you a bunch of info. “Basic” things like how long the protein is (# of amino acids) and how “big” it is (molecular weight, in Daltons (Da). It even tells you the number of atoms! It also tells you about how “basic” the protein is - in terms of how many “basic” (i.e. usually positively-charged) amino acids a protein has. This will impact the pI (isoelectric point) which is the pH at which the protein is neutral overall. Go below that pH and there are “excess” protons available, and the protein will be positively-charged on average. Go about that pH and there are “too few” protons available, so the protein will be negatively-charged. ProtParam tells you the theoretical pI which is super useful for doing charge-based protein purification (i.e. ion exchange chromatography). If your protein has a low pI, we say it’s “acidic” and usually use anion exchange. If your protein has a high pI, we say it’s “basic” and usually use cation exchange. more on all this here: https://bit.ly/isoelectricpoint & https://youtu.be/CLgzYBm_ymk and more ion exchange chromatography here: blog form: http://bit.ly/ionexchangechromatography ; YouTube: https://youtu.be/RGF1l572IZY It also gives you the extinction coefficient. WAY more on this here: http://bit.ly/bradforduv & http://bit.ly/proteinmeasuring but the key thing is it allows you to calculate a (pure) protein’s concentration based on how much UV light it absorbs. Extinction coefficients tell you the absorbance value (A) that corresponds to 10 mg/mL (1%) or 1 mg/mL (0.1%). Those percentages come from the weight/volume percentage convention that a 1% solution corresponds to 1 g/100 mL - more on why here: http://bit.ly/weightvolume & https://youtu.be/uo0Lx_OmKBA You can calculate the estimated extinction coefficient using free online software tools like Expasy ProtParam. I say estimated because context matters - the local environment around the absorbing part can influence how eager it is to absorb a photon When I do this for BSA I see this: Extinction coefficients: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient 42925 Abs 0.1% (=1 g/l) 0.638, assuming all pairs of Cys residues form cystines Ext. coefficient 40800 Abs 0.1% (=1 g/l) 0.607, assuming all Cys residues are reduced First it tells me the values under oxidizing conditions and below that it tells me the values under reducing conditions (the intracellular environment is reducing and we usually add reducing agents like DTT or β-mercaptoenthanol) to protein solutions to keep them happy outside the cell). It tells me this because cysteine crosslinks can also absorb, where applicable. Then I can plug this into Beer’s law (or have the computer do it for me) if I measure the absorbance. You measure this absorbance using something called a spectrophotometer. Basically it shines light through a solution and measures to what extent different wavelengths make it through (are transmitted) versus don’t make it through (are absorbed). This can be converted into concentration of solute (dissolved molecules) using Beer’s Law The equation is: A = εcl A = absorbance ε = extinction coefficient (aka molar absorptivity coefficient) - specific for particular molecule & particular wavelength; units of L mol-1cm-1 c = concentration (in mol/L) - this is molarity - a mole is just a chemist’s “baker’s dozen” - it’s Avogadro’s number (6.022 x 10^23) of something - solute molecules or donuts, it’s just a number http://bit.ly/solutionconcentrations l = path length (in cm) finished in comments