I take it that you want to check whether you protein remains folded in low pH? There are several possible ways to assess this:
1. Tryptophan fluorescence - if your protein has a buried tryptophan, you can record a fluorescence spectrum of your sample as you titrate the protein with an acid. The maximum emission wavelength of tryptophan changes upon solvent exposure (that is, when the protein unfolds).
2. CD spectroscopy - titrate your protein sample with a CD compatible acid (like hydrofluoric acid, be careful though) and record a CD spectrum as you decrease the pH. A major change in regular secondary structure content will reveal if (even partial) unfolding takes place. Be mindful that many salts and additives can be problematic in CD due to absorption and excluding these from your buffer solution might already influence stability.
3. SYPRO orange staining - The chemical SYPRO orange binds tightly to hydrophobic patches that are exposed when a protein unfolds. You can prepare a pH series of a protein solution on a PCR strip/plate and check if the fluorescence of SYPRO orange increases significantly in low pH values. You will need an RT-PCR machine with a suitable filter (like TAMRA) for conveniency.
4. SLS and DLS - Especially if your protein is oligomeric and susceptible to pH-induced dissociation, using these methods you can again assess its pH stability for instance in a 96-well plate format.
5. UV absorption - In principle you can measure how strongly your protein absorbs at 280 nm as a function of pH, taken that your protein contains aromatic residues. An unfolded protein in most cases absorbs more strongly than a folded one, although the absorption difference varies from protein to protein and in practice can be very small.
Additionally, if the acid itself is not enough to denaturate, you can use these methods together with temperature series or compounds that promote oligomer dissociation/denaturation (SDS, urea, guanidine HCl etc.) to check if the acidic environment weakens the stability of your protein. Most of these methods are rather sensitive and therefore do not require a lot of protein.
Hope this helps,
Arne
I take it that you want to check whether you protein remains folded in low pH? There are several possible ways to assess this:
1. Tryptophan fluorescence - if your protein has a buried tryptophan, you can record a fluorescence spectrum of your sample as you titrate the protein with an acid. The maximum emission wavelength of tryptophan changes upon solvent exposure (that is, when the protein unfolds).
2. CD spectroscopy - titrate your protein sample with a CD compatible acid (like hydrofluoric acid, be careful though) and record a CD spectrum as you decrease the pH. A major change in regular secondary structure content will reveal if (even partial) unfolding takes place. Be mindful that many salts and additives can be problematic in CD due to absorption and excluding these from your buffer solution might already influence stability.
3. SYPRO orange staining - The chemical SYPRO orange binds tightly to hydrophobic patches that are exposed when a protein unfolds. You can prepare a pH series of a protein solution on a PCR strip/plate and check if the fluorescence of SYPRO orange increases significantly in low pH values. You will need an RT-PCR machine with a suitable filter (like TAMRA) for conveniency.
4. SLS and DLS - Especially if your protein is oligomeric and susceptible to pH-induced dissociation, using these methods you can again assess its pH stability for instance in a 96-well plate format.
5. UV absorption - In principle you can measure how strongly your protein absorbs at 280 nm as a function of pH, taken that your protein contains aromatic residues. An unfolded protein in most cases absorbs more strongly than a folded one, although the absorption difference varies from protein to protein and in practice can be very small.
Additionally, if the acid itself is not enough to denaturate, you can use these methods together with temperature series or compounds that promote oligomer dissociation/denaturation (SDS, urea, guanidine HCl etc.) to check if the acidic environment weakens the stability of your protein. Most of these methods are rather sensitive and therefore do not require a lot of protein.
Hope this helps,
Arne
check the CD and fluorescence spectra of your protein at physiological pH and low pH. if they overlay, your protein's structure is acid resistant. Next check the activity of your protein at low pH by the standard activity assay (specific for your protein), if the activity is similar you can conclude that your protein is acid resistant. If it is not possible to do the activity assay at low pH, then after incubating the protein at low pH for sometime (30 min), dialyse the protein in physiological pH buffer and test the activity.
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