The Use of Steel-Tip Homogenizer Probe
Results in Sample
Carryover Contamination

Introduction

In any experiment involving different treatment groups or varying starting material it is important to ensure that no crossover or contamination of sample material occurs. We routinely work with mouse models of varying genotype and then study the effects of different treatments in these mice. More specifically, we measure the levels of apolipoprotein E protein and mRNA in brain tissue using a very sensitive and quantitative assay system. Therefore, we tested two probes for potential contamination between tissue samples during the homogenization process. The probes we tested were the plastic-tip disposable generator probe 04727-50 and the steel-tip probe 04727-69.

Procedure

All tissue was frozen in liquid nitrogen before homogenization. One half of the brain was homogenized with the plastic probe and the other half with the steel probe. The tissue was placed into 1 mL of guanidine buffer (5.0 M guanidine-HCL, 50 mM Tris pH 8.0) and allowed to thaw for 30 seconds before homogenizing on ice using Cole-Parmer PCR Homogenizer kit 04727-00. Each sample was homogenized for 45 seconds on low, medium, and high speeds in a 2-mL vial. The probes were then cleaned in water on high speed for two minutes, in homogenization buffer for 1 minute (twice), and wiped down with a Kimwipe (Kimberly-Clark). Homogenates were spun in an Eppendorf microcentrifuge at 14,000 rpm for 20 minutes at 4°C, and the supernatant assayed for protein content.

The micro BCA Protein Assay (Pierce) was used according to the manufacturer’s protocol. In brief, 150 µL of sample was pipetted into duplicate wells of a 96-well plate. 150 µL of working solution was added to each well, and the plate mixed for 30 seconds on a shaker. The plate was incubated at 37°C for 2 hours and the absorbance measured at 562 nm. To correct for different weights of tissue the following formula was used: Average protein concentration of wash/Average protein concentration of homogenate x 100%. These numbers were then used to compare the amount of protein carryover between the plastic- and steel-tip probes.

Results

Both the plastic-tip disposable generator probe 04727-50 and the steel-tip probe 04727-69 homogenized the brain tissue very efficiently. For comparison, we corrected for different starting amounts of protein by looking at the percent of wash homogenate protein compared to the total protein detected in the 1/2 brain homogenate. The plastic-tip disposable generator probe had a carryover of 0.6% of total protein from brain homogenate. The steel-tip probe had a carryover of 2.4% of total protein from brain homogenate.

It is worth noting that these numbers were obtained after processing of a single sample. Since the steel-tip probe would be used for all subsequent samples, we do not know if this carryover of protein contamination would accumulate over time. Extensive cleaning of the steel-tip probe between each sample may minimize this risk, however the technician time required for autoclaving and cleaning does not warrant use of the steel probe in our opinion. Also, SDS-solubilized tissue may show even more carryover, since a much larger fraction of the tissue is not soluble in SDS. Obviously, this is not an issue when using the disposable probes. For measuring small differences in mRNA between samples using an RT-PCR method, the plastic-tip disposable probes would clearly be preferred.