Correct Sample Prep for ELISAs and Activity Assays

To get optimum results correct sample prep for ELISAs and activity assays it is important that samples are prepared correctly.

Abcam’s suggested protocols for the preparation of cultured cell line and tissue samples for these experiments allow researchers to get the best data from their assays.

Choosing a Buffer for Cell Lysis

Cellular assays:

  • The lysis buffer provided with your kit should be used, for the majority of experiments this tends to be the assay buffer too. The lysis buffer may also include sodium vanadate and protease inhibitors. Protease inhibitors should be excluded for experiments requiring the detection of protease activity.
  • If no lysis buffer is provided, or if samples have been prepared following a different method, a PBS + 0.5% NP-40 (v/v) solution can be used. When using this solution, researchers should ensure that there will be no interference between the buffer and the components in their assay.
  • Sometimes a RIPA buffer can also be used for analyte detection. However, RIPA contains SDS, which interferes with the activity of proteins, and therefore enzymes, so it cannot be used to measure enzyme activity.

ELISAs:

  • The lysis buffer contained in the ELISA kit should be used.
  • If not available a RIPA buffer with added sodium vanadate and protease inhibitors should be used.

Procedure for Tissue Lysis

  1. The tissue sample should either be freshly collected or have been snap frozen on dry ice and then stored cryogenically (-80 °C).
  2. A small section of tissue, weighing a couple of grams, should be cut from the sample on dry ice. The section should be placed into a 5 mL universal tube and further cut into a collection of increasingly smaller pieces.
  3. 0.05 – 0.5 g of the tissue sample should be placed in a 1.5 mL homogenizer tube, e.g. a pre-loaded BeadBeater tube, on wet ice. Lysis buffer should then be used to fill the rest of the homogenizer tube.
  4. The sample should be homogenized in the tube for 90 seconds, then placed back onto ice.
  5. If the sample requires further homogenization repeat step four.
  6. The now homogenous sample should be centrifuged for 10 minutes at maximum speed and at 4 ° C.
  7. The supernatant should be collected for the assay. The supernatant should be kept on ice or, if it is to be used later, snap frozen.
  8. Protein concentration can then be measured using the BCA method. If a different buffer is used this should be ran as a negative control.

Protocol for Cultured Cell Lysis

  1. The cells should either be freshly collected or have been snap frozen on dry ice and then stored cryogenically (-80 °C).
  2. The pellets should be suspended in the chosen lysis buffer (50 - 100 μ L) and kept on ice for 10 minutes.
  3. The sample tubes should be placed in a water bath filled with ice-cold water and sonicated over three 50 second cycles - 30 seconds on, 10 seconds off, 10 seconds on.
  4. The sample tubes should be placed on ice for one minute, during which time the ice-cold water in the water bath should be changed.
  5. Step number three should be repeated.
  6. The samples should then be centrifuged for 10 minutes at maximum speed and at 4 ° C.
  7. The supernatant should be collected for the assay. The supernatant should be kept on ice or, if it is to be used later, snap frozen.

Components of the RIPA Buffer

  • 150 mM sodium chloride
  • 50 mM Tris pH 8.0
  • 0.5% sodium deoxycholate
  • 1.0% NP-40 or Triton X-100
  • 0.1% Sodium Dodecyl Sulphate (SDS)

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Apr 1, 2019 at 5:45 AM

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