FAQ: Protein Carbonyl Spectrophotometric Assay

Q: Is there an advantage to using the Protein Carbonyl ELISA Kit compared to the spectrophotometric format?

A: Our Protein Carbonyl ELISA kit is about 5-10 times more sensitive than our Protein Carbonyl Spectrophotometric Assay and only requires 1 µg of protein per assay versus 250 µg protein per assay with the spectrophotometric format.  Many more samples can be processed at once in the ELISA kit, while the spectrophotometric kit is performed in individual tubes. The spectrophotometric kit format is designed for use by laboratories without an ELISA plate reader.

 

Q: Is this kit species specific?

A: Because the structure of protein carbonyl group is the same regardless where the sample is from, these protein carbonyl kits are NOT species specific and can be used for any protein samples. 

 

Q: What lysis buffer do you recommend?

A: RIPA buffer or any other detergent based cell lysis buffer can be used to lyse cells.  Here is the lysis buffer we use in the lab: 50 mM Tris-HCl, pH 7.5, including 150 mM NaCl, 1 mM 2-glycerophosphate, 1% Triton X-100 (or 1 % NP-40), 1 mM EDTA, 1 mM EGTA and a proteinase inhibitor cocktail.
 

Q: Is the protein concentration measured twice?

A: Yes, the protein concentration is measured before starting the assay and again during the assay in step 5.  The second measurement is important because by this point in the assay the protein samples have been through a TCA precipitation and not all of the protein that was started with may be dissolved.

 

Q: What tubes should I use for this assay that can accommodate 2.5ml?

A: You may use a 15 mL conical tube for steps 1 and 2 and spin in a centrifuge.  If you only have access to a microcentrifuge, an option is to assemble the solutions in a 15 ml conical and then split the volume into multiple microcentrifuge tubes for the spin.  During the wash in step 3, the two pellets for each sample can then be combined into a single microcentrifuge tube.