I am conducting research on urinary protein biomarkers. I am facing a few issues that I would like to share in the hope that someone can help me to overcome them.
1. Protein solubilization
a) I am using phosphate buffer saline (PBS) pH 8.0 to solubilize the proteins. However, after I have reconstituted the pellets, they are not completely soluble in the buffer. How can I solubilize the entire protein?
b) I am extracting the proteins with ammonium sulfate (by salting out). I then perform SDS electrophoresis. The resolution of the gels is not good enough to qualify the matching software, as the bands appear either very dark and diffuse or interfere with the next sample (as shown in the attached Figure 1). Why is this?
c) There is a line above the protein migration as shown in Figure 2. What is the reason for this?
2. Protein quantification
I am using the Lowry assay from to quantify the proteins. However, sometimes the readings are not accurate. What is the most sensitive assay for protein quantification?
I would be grateful for any help that you can provide.
Thank you for your time
1. Since you are going to run the proteins on SDS-PAGE anyway, you should be able to dissolve them directly with SDS-PAGE sample buffer. Or, if you don't want to do that, you could just use 1% SDS. Quantitation of protein dissolved in SDS-PAGE sample buffer is difficult, but if you use SDS alone, you can use the BCA assay for quantitation.
Another alternative for dissolving insoluble proteins is to use 6 or 8 M urea.
2. Precipitating the proteins with ammonium sulfate creates a problem for SDS-PAGE. The pelleted protein will contain a high concentration of entrapped ammonium sulfate. The high ionic strength of the redissolved protein sample will cause the bands on SDS-PAGE to migrate sideways if the adjacent bands have lower ionic strength. Try to avoid ammonium sulfate precipitation if you can. If not, you could dialyze the samples to remove the residual ammonium sulfate.
I would judge your gel to be overloaded, which is causing some of the poor migration quality. Load a smaller amount of protein. It also helps to run the gels at a lower voltage for a longer time to sharpen the bands. Use 2/3 to 3/4 of the recommended voltage.
3. The Lowry, BCA and Bradford assays have similar sensitivities. The Bradford assay is not compatible with high detergent concentrations. The BCA assay is not compatible with reducing agents (mercaptoethanol, dithiothreitol, TCEP). I have found that the sensitivity of the Bradford assay is very high when using the Bradford reagent supplied by Bio-Rad. I use a standard curve of 0 - 2.75 µg BSA in 0.25 µg increments (10 µl sample) in a 96-well plate format with 200 µl of reagent, and the signal is very strong.
The sensitivity of the Lowry assay can be increased by precipitating a large volume of the protein with TCA and sodium deoxycholate. To 1 ml of a dilute protein solution, add 100 µl of 72% (w/v) TCA and 100 µl of 0.15% sodium deoxycholate. Incubate on ice for 10 min, then centrifuge at maximum speed for 10 min. Remove the supernatant very thoroughly by gentle suction, being careful not to disturb the pellet. The Lowry assay is then performed in the same tube.
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