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Detergents Physical and Chemical Properties
Dialyzable Detergents

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Protein Protocol: Proteins were diluted to 0.01 mg/ml and 0.1 mg/ml in glycine pH 2.8; PBS pH 7.2; or sodium bicarb, pH 9.2.
50 µl of each protein solution was added to a 10K MINI unit and dialyzed overnight against PBS, pH 7.2. The recovered sample (~50 µl) was put in a microwell plate and mixed with 100 µl of Micro BCA™ Protein Assay Reagent.

Dialysis Rate and Sample Recovery

The 3.5K Slide-A-Lyzer MINI Dialysis Unit was used for salt reduction analysis. Sample volumes in the range of 5-100 µl of 1 M NaCl were placed in the Slide-A-Lyzer MINI Dialysis Unit and dialyzed against 1 liter of water for 10 minutes. To recover the smallest (5 µl and 10 µl) volumes from the Slide-A-Lyzer MINI Dialysis Unit, the device was gently tapped on the bottom edge to collect the sample in a corner prior to pipette recovery. NaCl standards and samples were diluted in 50 ml Milli-Q water and read with a conductivity meter (Cole-Parmer). Figure 3 shows the residual percent of NaCl remaining for each volume. Dialysis rate per time of 100 µl 5 M NaCl was also analyzed by conductivity. The results are shown in Figure 4. The rate of pH exchange in the Slide-A-Lyzer MINI Dialysis Unit is also very rapid. In less than 10 minutes, 100 µl of IgG elution buffer, pH 2.8 is converted to pH 9.5 from dialysis against 1 liter of BupH Bicarbonate, pH 9.4.

Figure 1. After dialysis against water for 10 minutes, the percent residual 1M NaCl is 0% for 5-10 µl samples, < 20% for 20-50 µl samples, and < 40% for 60-100 µl samples.

Figure 2. Dialysis time course for 100 µl of 5 M NaCl in a Slide-A-Lyzer MINI Dialysis Unit.

• Improved electrophoretic resolution
• Agarose electrophoresis
  

  Previews (2001.) New Slide-A-Lyzer MINI Dialysis Unit trial sizes. 5(3), 6.

• Capillary electrophoresis Sykaluk, L., Brennan, T., King-Spengler, T. and McKibben, S. (1998). Slide-A-Lyzer MINI Dialysis Units for microliter samples, Previews 2(4), 13.
• Improved restriction digests

B.

Figure 3. Gel capillary electrophoretic analysis of a rhodamine-oligonucleotide (A) prior to salt reduction and (B) after salt reduction using a Slide-A-Lyzer MINI Dialysis Unit.

Brown, T.A., ed. (1991). Molecular Biology LabFax, BIOS Scientific Publishers Limited: Oxford, UK, p. 116.

• Improved electrophoretic resolution
  • Reduction of salt in sample
  • Reduction of buffer concentration or components
• Improved conjugation pH 7 vs. pH 9.5
• Improved protein digest (i.e., salt, organics, detergents, pH and denaturants alter protein folding and thus cleavage with reductants, enzymes and other chemical cleaving agents)

Microconjugations

Often, only small amounts of sample are available or needed for assay evaluation. There are many commercially available activated enzymes and compounds to generate probes. The Slide-A-Lyzer MINI Dialysis Unit is the ideal tool for buffer exchange, for serving as a reaction vessel or for cleaning up the probe. Antibody-enzyme probes were prepared with EZ-Link Plus Activated Peroxidase, an aldehyde-activated horseradish peroxidase (HRP) that can be coupled to primary amines on antibodies. In our experience, high pH coupling provides higher activity probes using this chemistry. We used the 10K Slide-A-Lyzer MINI Dialysis Unit to dialyze 100 µl of 2.4 mg/ml goat anti-mouse IgG against 1 liter of BupH Bicarbonate, pH 9.4 or BupH PBS, pH 7.2 for 1 hour at room temperature (RT). At 1 hour, a 50 µl aliquot of 10 mg/ml EZ-Link Plus Activated Peroxidase was added to the dialyzed antibodies to create an 8 M excess of enzyme to antibody. Dialysis continued for 2 hours. Then, 1 µl of 5 M NaBH₃CN was added to reduce Schiff's base for 30 minutes. After reduction, a 5 µl aliquot of 3 M ethanolamine was added to quench any unreacted sites for 30 minutes. Samples were then dialyzed overnight against 1 liter of fresh BupH PBS. Figure 4 shows the results of a direct ELISA measured using a Molecular Devices plate reader. The high pH coupling protocol resulted in a higher activity probe. We also successfully produced antibody-enzyme conjugates using the Slide-A-Lyzer MINI Dialysis Unit and alternative coupling chemistries.

Figure 4. Conjugates prepared in the Slide-A-Lyzer MINI Dialysis Unit were added at 100 ng/well to a BSA-blocked, mouse IgG-coated plate for 2 hours at 37°C. The plates were washed with PBS containing 0.05% Tween-20. ABTS substrate (100 ml) was added to each well to detect the HRP conjugate presence. The conjugate prepared at pH 9.5 exhibited greater activity than the pH 7 conjugate.