Preliminary Test of IgG Binding to Protein A Beads

Last week, Barrett and I made up the three buffers needed to perform this test:

Dissociating buffer: 0.1 M glycine-HCl, pH 3.5

1 M Tris

Binding buffer: 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5

In addition, I made a solution of protein A beads that consists of 0.0166 g of beads in 50 mL of DI water. I put it in the refrigerator to swell. At the end of the day, Barrett added ethanol to make a 20% ethanol solution so nothing would grow in the solution over the weekend.

Check to make sure that the Bradford assay detects the protein.

• For this experiment, we used goat anti-mouse. The literature we have doesn’t have any information about the binding strength of goat proteins (and the protein we have is about 6 years old) but we thought we could use it as a first trial step.
• We just did an eye test, but the Bradford dye turned a bright blue when added to a sample of goat protein when it is normally a pale brown, so we know the the dye is recognizing the protein.

Remove the ethanol:

• Use centrifuge to spin down the beads (1200 RPM for 5 minutes)
• Use a 1 mL pipetter to remove most the supernatant (ethanol)
• Break up the pellet, suspending the beads in the small amount of ethanol left (pump with pipetter)
• Move solution to a 1.5 mL centrifuge tube
• Add 1 mL of binding buffer to the original tube, shake so the leftover beads will become suspended
• Transfer to 1.5 mL tube

Rinse off binding buffer:

• Spin down the beads (1000 RPM for 5 minutes)
• Use pipetter to remove most of the supernatant
• Add the elution buffer; mix well

Pre-elution step:

• Spin down beads
• Remove supernatant
• Add dissociation buffer to get rid of any remaining antibody

Equilibrating step:

• Spin down beads
• Remove supernatant
• Add binding buffer

–> Repeat this step twice

To make sure that it is equilibrated:

• Transfer bead solution to a 15 mL tube and add 3 mL of binding buffer (this ensures that we aren’t accidentally removing some of the beads, which happens at low volumes)

Add Goat IgG:

• Spin down
• Remove supernatant
• Pump with pipette
• Transfer back to a 1.5 mL centrifuge tube
• Add 1 mL of binding buffer to the 15 mL tube to suspend the remaining beads; transfer to 1.5 mL centrifuge tube
• Add 200 μL of Goat IgG (we don’t know the concentration, but that should be more than enough to bind)
• Mix well

Take samples and wash with binding buffer until absorbance at 280 nm is less than 0.02

• We rinsed with the binding buffer five times (for a total of six samples)

Add dissociating buffer.

Does the protein come off the beads? Use bradford assay to find out.

Use TECAN to measure absorbance at 595 nm (for bradford assay):

Finishing steps:

• Put bead solution into a 15 mL tube, add an excess of binding buffer to neutralize pH.
• Spin down and remove supernatant.
• Put beads into 4 mL of binding buffer
• Add 1 mL of 100% ethanol to make a 20% ethanol storing solution.

Conclusions:

Using the TECAN, we looked at the absorbances and they seem to hover just about the absorbance of the buffer on its own. We believe that this is quite promising considering that we were working with proteins that were kept in the refrigerator (instead of the freezer) for six years and considering that we don’t know the binding strength of goat IgG. The next step is to purchase fresh protein A and human IgG (which is what we will be using in future experiments) so we we can perform better tests.