Getting BSA Through Membranes

This is a follow-up post to “Protein Assay Test.” I realized that I never posted the final stag of my experiments, in which I finally got BSA to go through the membranes.

In “Protein Assay Test,” I described several experiments that I set up to see if I could get BSA to pass through a membrane. I use a Bio-Rad Assay to test for BSA in the filtrate. A higher absorption (measured with the TECAN) means a higher concentration of BSA. At the end of “Protein Assay Test,” I had done an experiment with 10 mg/ml BSA (in HBSS) and membranes with pinholes where I was getting absorptions that suggested that BSA was passing through. Here are the results of further experiments I conducted, to verify that I was actually getting BSA to pass through a membrane.

7/7/09: Set Up Protein Assay, Take Two (to see if I could replicate the results I got with the previous experiment)

Set-up: SepCon in a 24-well plate, 200μl in apical, 600μl in basolateral

Wafer: 306

2 SepCons:

1. 10 mg/ml BSA in HBSS, membrane with pinholes
2. just HBSS, membrane with pinholes

7/8/09: Set Up Small-Scale Protein Assay (small basolateral volume, so will have a higher end concentration of BSA)

Set-up: SepCon in a 1.5ml centrifuge tube, 200μl in apical, 60μl in basolateral

Wafer: 306

1 SepCon:

1. 10 mg/ml BSA in HBSS

7/9/09: When I checked both of the above experiments, the membranes had all broken, so I set up both experiments again but I used a different wafer.

Wafer: 222 (has comparatively large pores)

Regular set-up:

1. 10 mg/ml BSA in HBSS, membrane with pinholes
2. just HBSS, membrane with pinholes

Small-scale set-up:

1. 10 mg/ml BSA in HBSS, membrane without pinholes

7/10/09: Results (after 24 hours)

	Absorption
	sample 1	sample 2
HBSS:	0.2055	0.2067
BSA:	0.577	0.5913
small-scale BSA:	0.9083

7/13/09: Since the experiments worked with BSA in HBSS, I tried BSA in PBS again.

Wafer: 222

Regular set-up:

1. 10 mg/ml BSA in PBS, membrane with pinholes

Small-scale set-up:

1. 10 mg/ml BSA in PBSS, membrane with pinholes

7/14/09: Results (after 24 hours)

	Absorption
	sample 1	sample 2	sample 3
PBS:	0.216
BSA:	0.5101	0.4787	0.5789
small-scale BSA:	0.8858

7/16/09: Membranes without pinholes

Wafer: 222

Regular set-up:

1. 10 mg/ml BSA in PBS, membrane without pinholes

Small-scale set-up:

1. 10 mg/ml BSA in PBSS, membrane without pinholes

7/17/09: Results (after 24 hours)

	Absorption
	sample 1	sample 2
PBS:	0.2245	0.2223
BSA:	0.4292	0.45
small-scale BSA:	0.9963	1.0245

7/20/09: Results (after 4 days)

	Absorption
	sample 1	sample 2
PBS:	0.1922	0.1973
BSA:	0.7949
small-scale BSA:	1.0691

7/22/09: Results (after 5.5 days)

	Absorption
	sample 1	sample 2
PBS:	0.2073	0.2008
BSA:	0.8019	0.8512
small-scale BSA:	1.0274	1.04

In conclusion, BSA went through wafer 222.