Manual / Product Insert

User Guide: Protein Precipitation Plates

Version: Jan. 2015

Product FAQ

I'm seeing protein precipitation during native binding when using the ProBond Purification System. What do you suggest I do to avoid this?

Answer

Please see our suggestions below:

-Add 0.1% Triton X-100 or Tween-20 to help solubilize further; purify at room temperature if protein is not temperature sensitive. However, keep in mind that most proteins are temperature sensitive.
-If secreted proteins are in media with low pH, they must be dialyzed to avoid Ni2+ reduction.
-If solubility is a real problem (e.g., microsomes), include up to 0.2% Sarkosyl in the 6 M guanidine lysis buffer; this will help to solubilize everything and may be still compatible with the ProBond or Ni-NTA purification column.

Answer Id: E12980

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Product Literature

Application Brief: HyperSep Protein Precipitation Plates 60305-201

Product FAQ

How can I precipitate proteins that are in solution at very low concentrations?

Answer

The following protocol can be used to precipitate small amounts of protein:
(1) Add 1/100 vol. of 2% Na deoxycholate (DOC) to one volume of protein solution. Vortex and let stand for 30 min at 4 degrees C.
(2) Add 1/10 vol of 100% trichloroacetic acid (TCA), vortex and let stand overnight at 4 degrees C.
(3) Spin in a microcentrifuge at maximum speed for 15 min at 4 degrees C. Carefully discharge supernatant and retain the pellet.
(4) Dry tube by inverting it on tissue paper (pellet may be difficult to see). [OPTION: Wash pellet twice with one volume of cold acetone (acetone keep at -20 degrees C). Vortex and re-pellet samples at max speed for 5 min between washes].
(5) Dry in a vacuum centrifuge (e.g., SpeedVac system ) or air dry.
(6) For SDS-PAGE, resuspend samples in a minimal volume of sample buffer.
Note: The presence of some TCA can give a yellow color as a consequence of the acidification of the sample buffer; titrate with 1 N NaOH or 1 M Tris HCl pH 8.5 to obtain the normal blue sample buffer color.

To prepare 100% TCA: dissolve 1 kg of TCA in 454 mL water. Maintain in a dark bottle at 4 degrees C.

Answer Id: E4455

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Product FAQ

With the Thermo Scientific Coomassie (Bradford) Protein Assay, How can interfering agents be removed?

Answer

The Thermo Scientific Coomassie (Bradford) Protein Assay Kit is incompatible with high detergent concentrations. This can be overcome by removing the detergents from the protein solution. The Thermo Scientific Detergent Removal Resin can be used to remove the detergents from protein solutions. Other methods for removing detergents include size exclusion, dialysis, or protein precipitation.

Answer Id: E13422

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Product FAQ

A component of my lysis buffer is known to interfere with my protein assay of choice. How can I remove it?

Answer

Several strategies exist for overcoming or eliminating sample incompatibility with protein assays. The simplest method is to assay the sample after diluting it several-fold in a compatible buffer. If the starting concentration of protein is sufficient to remain within the protein assay working range upon its dilution, then this method will often reduce the amount of interfering substance in the sample to the point where it no longer interferes. Another method is to dialyze or desalt samples into a buffer that is compatible with the assay.

Precipitation can be used to eliminate interfering substances. After causing the protein to precipitate with acetone or trichloroacetic acid (TCA), the supernatant containing the interfering substance can be removed. Then the protein pellet is dissolved in the assay working reagent, and the protein assayed performed as usual. A general protocol for protein precipitation is provided in our Tech Tip

Answer Id: E15578

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Product FAQ

Can I re-use diluted BSA standards for subsequent protein assays?

Answer

The challenge of diluted BSA standards is that they need to be protected from microbial growth, evaporation and protein precipitation. These will result in artificially high or low protein concentration values. Including additional sodium azide with the diluted protein standards to maintain a final concentration of 0.05% will help prevent microbial growth. The shelf life of diluted standards can be extended if they are sealed and kept refrigerated.

Answer Id: E13299

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Product Literature

Tech Tip: Acetone precipitation of proteins

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