Sapidyne Instruments

Newsletter #10

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Cell Pellets & the Effects on Equilibrium

KinExA affinity measurements for a specific binding target to cell expressed receptors require the reaction to be at equilibrium. After cells equilibrate with the target, they are pelleted and the supernatant is retained for subsequent experiments (Figure 1).

During the centrifugation process, which takes a few minutes, the cells become concentrated into a pellet at the bottom of the sample tube. This process will not effect the equilibrium of the samples and can be demonstrated by following the equations.

When the cells are pelleted, their concentration is increased in a local area, but the free target concentration did not change. Since both the bound and unbound cell concentrations are increased by the same amount in the pellet, the ratio [R]/[RT] stays the same. Looking again at Equation 2, you can see that although the local concentration of the cells changed, the overall equation does not. Therefore, during the pelleting process, you can be sure the equilibrium does not shift.

In fact, after the cells are pelleted, you can remove the supernatant and the equilibrium is still not affected. Although you did remove some of the volume, you did not change the free target concentration therefore everything in Equation 2 remains the same.

Kinetic Exclusion Assay (KinExA) Now On Wikipedia: en.wikipedia.org/wiki/Kinetic_exclusion_assay

Advanced Flow Cells for Sticky Systems

At Sapidyne, our R&D team is continuously working to improve our measurement capabilities and overcome the issues that interfere with getting consistent, clean data. One of these issues is measuring “sticky” systems that cause baseline creep and titrant related non-specific binding. See Tech Note TN216 Baseline Creep and Tech Note TN210 Titrant Related NSB for more information. In our 2013 Newsletter we introduced our siliconized Flow Cells to help fight these sticky systems. We now have a new, improved, flow cell that does the job better.

The inner surface of the new flow cell has been chemically modified by attaching a PEG-Silane molecule. This makes a hydrophilic surface thereby reducing the ability of proteins to stick. We tested a particularly sticky system of antibodies extracted from chicken yolks that were run through the Hydrophilic Flow Cell. The Hydrophilic Flow cell resulted in only 0.30 V of baseline creep after 10 runs compared to the 0.77 V increase observed in the Siliconized Flow Cell.

Available Flow Cells can be seen in Figure 2 and differentiated by exit line color. See Table 1 for more information and contact Sapidyne for ordering information.

Flow Cell options: Non-Treated (Blue line) – Part #392534 (3200) / #392360 (4000), Pack of 3. Siliconized (Black line) – Part #392151 (3200) / #392170 (4000), Single. Hydrophilic (Green line) – Part #392534 (3200) / #392544 (4000), Pack of 3.

 

LED Lamp Replacement – KinExA 3200

We are now offering an LED lamp replacement with a much longer life span and a 10 year warranty (Figure 3). The LED lamp is a direct replacement for the halogen lamp (Figure 4).

Advantages to using the LED lamp are: fewer lamp replacements, lower baseline signal, and low risk of lamp burnout while an experiment is running. LED lamps also use 10% of the electricity that halogen lamps use.

Replacing your old lamp with the new LED lamp is straight forward and can be completed in 5 minutes with 3 easy steps:

  1. Turn the old lamp off and remove it from the instrument.
  2. Plug in the new LED lamp and turn it on.
  3. Adjust the new LED lamp to ensure proper focus and alignment.

An in-depth How to Guide can be found on our website under the resources tab titled HG204 Lamp Replacement and Alignment, which walks you through the alignment process in detail with helpful pictures to guide you. There are specific LED lamps depending on the filter set being used. Refer to Table 2 for LED lamp specifications.

LED Lamp options: 3200 LED Lamp, Red (Part #511100) – Red Filter. 3200 LED Lamp, Blue (Part #511101) – Blue Filter. 3200 LED Lamp, Green (Part #511102) – Green Filter.