Hello!
I'm purifying a protein and have several questions about what is appropriate to include in the extract solvent at different stages of the purification and characterization phases.
(1) The protein solvent contains 1% Triton X-100 and likely contains some NaCl as I am using 200 mM NaCl for elution during anion exchange chromatography. Can I include the Triton X-100 and residual NaCl in the protein solvent during Bradford assay? I initially thought this might be okay since I'm using a small amount of protein solution (2 microliters) relative to the ~1 mL sample of Bradford reagent and buffer, but am unsure if this might interfere with my results. Can I include them in the sample solvent or should I remove the salt (dialysis) and Triton X-100 (on a spin column)?
(2) Similar to the situation described above, can I include in the NaCl and Triton x-100 in the protein extract solvent during SDS-PAGE? Since I don't yet know how the concentration of my extract I'm not sure what the ratio of protein extract to SDS-PAGE sample buffer is yet.
High salt will prevent proper migration of your sample in the gel. shouldn't interfere with bradford unless salt concentration is very high
Triton will affect binding of bradford to proteins (unless the concentration is extremely low), and so interfere with the assay. it will not interefere with SDS-PAGE.
The salt should not interfere with either Bradford assay or SDS-PAGE; also, it seems that your protein is sufficiently concentrated (you mentioned using 2 ul in Bradford assay) so you will not introduce much salt in either of the assays anyway.
The only issue is Bradford-Triton x-100 incompatibility.
However, if you can order it there is a commercial version from Pierce that allows you to use Triton in your measurements https://www.thermofisher.com/order/catalog/product/23246S#/23246S
Good luck!
(1) The protein solvent contains 1% Triton X-100 and likely contains some NaCl as I am using 200 mM NaCl for elution during anion exchange chromatography. Can I include the Triton X-100 and residual NaCl in the protein solvent during Bradford assay? I initially thought this might be okay since I'm using a small amount of protein solution (2 microliters) relative to the ~1 mL sample of Bradford reagent and buffer, but am unsure if this might interfere with my results. Can I include them in the sample solvent or should I remove the salt (dialysis) and Triton X-100 (on a spin column)?
Some comments:
Bradford´s method is just an approximation of protein concentration (unless you have a pure protein and construct a calibration curve with the same protein). Thus do not expect to much from it. Most surfactants interact with Bradford´s dye and change the absorption behavior. Do not know what is going to happen with your particular case. What you can do is add triton and salt to the standards at the same concentration of the samples. Finally, mixing 2 ul with 1 mL will end up in huge errors.
(2) Similar to the situation described above, can I include in the NaCl and Triton x-100 in the protein extract solvent during SDS-PAGE? Since I don't yet know how the concentration of my extract I'm not sure what the ratio of protein extract to SDS-PAGE sample buffer is yet.
Some comments:
I have run samples with 1 M NaCl with no problems. Not sure about the surfactant as it will compete for protein binding with SDS. Run the sample and you will see. With respect to sample loading and mixing with sample buffer, do as I do: use undiluted sample, 10 times diluted, and 100 times diluted and run the electrophoresis.
- In general, dye-binding assays (including Bradford) are incompatible with detergents, as those interfere with dye-protein interaction. Very small concentration (
Actually, removal of detergents by dialysis (or ultrafiltration) is usually difficult, because of micelle formation. The actual concentration of the free detergent is the cmc, often in the µM - mM range. If the activity of the protein is not important, then the chloroform/methanol precipitation is usually the simplest method of detergent removal. If the protein needs to be active, then detergent-binding gels like BioBeads SM2 (doi:10.1016/0003-2697(73)90436-3) or XAD-resins are the method of choice.
Short answer: no. Practically Triton will not interfere at low concentration. Make sure you dilute your standards in your Triton buffer. As for SDS PAGE, Triton will not affect your migration provided your have enough SDS in your sample buffer and your Triton is below, ballpark, 0.1% final. As for salt, again, stay below 0.5M final and you're safe.
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