Denaturing Cell Extraction Buffer

We have 5 different cell and tissue extraction buffers suitable for preparing mouse cell and tissue extracts. These buffers can be used to extract cells and tissues from many other species as well. The exact compositions of all of our buffers are proprietary, but they are similar to those described by many researchers.

Four of these buffers can be used to prepare extracts which can be analyzed with our ELISA and Luminex kits and by Western blotting. Our Cell Extraction Buffer (FNN0011) contains extra phosphatase inhibitors and resembles the RIPA formulation that many people use. Our Tissue Extraction Reagents I (FNN0071) and II (FNN0081) contain different concentrations of NaCl and different surfactants, but are otherwise similar to each other. For those who prefer using an extraction buffer containing the detergent NP-40, we have our NP-40 Lysis Buffer (FNN0021). Finally, we sell a Denaturing Cell Extraction buffer (FNN0091) which contains 3 detergents and a chaotropic agent. Extracts prepared with FNN0091 can be analyzed with our ELISA kits and by Western blotting only. These buffers do not contain protease inhibitors, which the investigator should add right before use.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Here are possible causes and solutions for this issue:

- Check the protocol settings (make sure you select the correct DD settings).
- Check the level of sheath fluid and empty the waste.
- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are possible causes and solutions for this issue:

This usually indicates that the beads have been photobleached. This problem can also be caused by exposing the beads to organic solvents. Unfortunately, the assay will have to be repeated because the beads cannot be restored. The beads must be protected from light and organic solvents.
Alternatively, the instrument may be off in its measurements or you may have a calibration issue. Call the manufacturer for a service appointment.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

This indicates that an incorrect buffer was used for the final step. The Wash Solution provided in the kit must be used for washing the beads and the Reading Buffer should be used for resuspending the beads before loading them into the Luminex instrument. The osmolarity of the solution will impact the size of the bead, and any change in the bead size will alter detection by the instrument.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are some suggestions:

- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.
- Review the instrument settings and make sure they are appropriate for the assay being run (adjustment of needle height, make sure you select the correct bead gates and the correct DD settings).
- Shake the plate before acquisition on the instrument to resuspend the beads.
- Vortex the beads for 30 sec before adding them into the plate.
- Washing: Do not forget to keep the plate for about 2 mins on the Hand-Held Magnetic Plate Washer before emptying the plate.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.