Test of Theory– Can we successfully bind Human IgG to the protein A beads and then remove it with buffer B?

Test of Binding/ Unbinding (Carrie and Barrett): Test to see if we can successfully bind Human IgG to the protein A beads and then remove it with buffer B.

After ordering the new human IgG and protein A beads, I have been working on a higher-quality analysis of the binding and dissociating of the IgG and the beads.

Use “Protein A Beads and IgG Binding and Filtering (with SepCon Format)”

9/23/09: Swell Protein A Beads in Buffer A. Add Human IgG.

Swell Beads:

• Added 200μl of buffer A. The mixture was very dry.
• Added an additional 200μl, but it was still very dry.
• Added 200μl more. The mixture was a slurry, but a very thick one with the beads crusting onto the walls and cap of the conical.
• Added 400μl. The slurry was still very thick and the beads were not staying in solution
• Add more buffer. Total volume= 11 ml.

Store in 20% ethanol.

9/24/09

1) Rinse beads with Buffer A to remove ethanol.

2) Add human IgG to the beads.

9/25/09

1) Rinse with Buffer A to get rid of any unbound IgG (rinse until obtain a neutral Bradford Assay reading).

Bradford Assay:

• 2×10μl blank samples (Buffer A) [B1&2]
• 2×10μl samples after initial spin-down (unbound IgG) [B3&4]
• 2×10μl after 1 rinse [B5&6]
• 2×10μl after 2 rinse2 [B7&8]
• 2×10μl after 3 rinses [B9&10] (B7 had a few beads in solution)

–> All appear light brown. This is good because it means that there is very little unbound IgG in solution.

2) Try to unbind IgG from beads with buffer B.

• Add 1mL of buffer B (at pH 4). Mix for 15 minutes at room temperature; spin for 2 minutes at 1500 rpm.
• Add 59 μl of 1M NaOH, to neutralize the pH of buffer B.
• Take 2×10μl for protein assay [A5&A6] –> When add dye, well is light brown. If the IgG had come off the beads, it should be blue.

3) Try to unbind IgG again.

• Add 1mL of buffer B. Mix for 15 minutes at room temperature; spin for 2 minutes at 1500 rpm.
• Transfer 1 mL to a new tube and add 59μl of 1M NaOH.
• Take 2×10μl for protein assay [A7&8]. –> Still seems like no IgG is coming off.
• [A9&10] –> Buffer B, to have a blank to compare the other results to.

4) Try again, but mix for 30 minutes.

• Add 1mL of buffer B. Mix for 30 minutes at room temperature; spin for 2 minutes at 1500 rpm.
• Transfer 1 mL to a new tube and add 59μl of 1M NaOH.
• Take 2×10μl for protein assay [C1&2].

–> Still seems like no IgG is coming off

5) New attempt: Make buffer B at pH 3 and repeat elution [C3&4]; mix for 30 minutes.

• It worked!!!!
• [B11&12]–> New Buffer B (blank)

6) Finishing steps:

• Do 1 more elution to see if any more IgG–> Still IgG
• Do 1 last elution (2 hours) to remove all of IgG from beads
• Take 2×10μl sample [D1&2]
• Do protein assay
• Still a bit high ABS
• Rinse beads in 20 RV of Buffer A (~7 spins of 1.5mL)
• Store beads in 20% ethanol/ 80% Buffer A

TECAN readings:

<>	1	2	3	4	5	6	7	8	9	10
A	blank (buffer A)		After protein A bead+ IgG spun down		after 1 rinse with Buffer A		after 2 rinses with Buffer A		after 3 rinses with Buffer A
B	0.2113	0.2157	0.2183	0.209	0.2189	0.2077	0.2102	0.2103	0.2484	0.2015
C
	Elution
	Buffer B		Elution 1		Elution 2		Elution 3
	0.2095	0.2027	0.2429	0.2492	0.2088	0.2039	0.2338	0.2238
	Change to pH 3 elution buffer
	new Buffer B		Elution 1		Elution 2		2 Hour Elution
	0.1963	0.2019	0.3637	0.3754	0.2968	0.3022	0.2309	0.2367