Correct sample preparation is a key step for optimal performance for ELISAs and activity assays.
Our recommendations for tissue and cultured cell line preparation for ELISAs and activity assays will help you get the best from your assays.
Buffer selection for lysis of tissues and cells:
For ELISAs:
Use the suggested lysis buffer provided with your kit or RIPA buffer supplemented with protease inhibitors and sodium vanadate.
For cellular assays:
Use the suggested lysis buffer provided with your kit, which, in most cases will be the assay buffer itself. Lysis buffer can be supplemented with protease inhibitors and sodium vanadate, except for those assays where you want to detect activity of a protease.
Alternatively, a PBS + 0.5% NP-40 (v/v) solution could be used for lysis if no lysis buffer is provided or if samples have been prepared for other experiments. In this case, however, you should check the compatibility of the components to ensure that they will not interfere with the assay.
RIPA buffer can also be used occasionally for detection of analytes. RIPA buffer is not compatible with assays that quantify enzyme activity as the SDS interferes with the protein activity.
Procedure for lysis of tissue:
Use freshly collected tissue or tissue that has been snap frozen on dry ice and stored at -80°C. On dry ice, cut a small piece of tissue with a scalpel (a few grams) and transfer to a 5 mL universal tube. Continue to cut this small piece of tissue into even smaller pieces with the scalpel.
Place 0.05 - 0.5 g of tissue into a 1.5 mL homogenizer tube e.g. BeadBeater tube (pre-loaded with glass beads) on wet ice. Fill up the homogenizer tube with lysis buffer.
Homogenize the sample in the homogenizer tube for 90 seconds, then place on ice again.
If the sample is not completely homogenized, repeat step 3.
Centrifuge the homogenized sample at maximum speed for 10 minutes at 4°C.
Harvest the supernatant for the assay. Keep the supernatant on ice (or snap freeze) until ready to use.
Use the BCA method to determine the protein concentration. If a new buffer has been used, run that as a negative control.
Procedure for lysis of cultured cells:
Use freshly collected cells or cell pellets that have been snap frozen on dry ice and stored at -80°C.
Resuspend the pellets in > 50 - 100 μL of desired lysis buffer. Leave on ice for 10 minutes.
Sonicate the samples in an ice cold water filled vessel for 3 x 50 second cycles ( 30 seconds on, 10 seconds off, 10 seconds on).
Place tubes on ice for 1 minute whilst the water bath is refilled with ice cold water.
Repeat the sonication step again.
Centrifuge the samples at maximum speed for 10 minutes at 4°C.
Harvest the supernatant for the assay. Keep the supernatant on ice (or snap freeze) until ready to use.