Section: 802, (Thursday)

Group: 4

Names: John Gibson, Kyle LaPlant

Instructor: S. Royan, PhD

Date: Nov 14, 2024

University Of Massachusetts,

Lowell, Fall 2024

Immunology Lab Report 5: Bio-Rad Microassay for Immunoglobulin G Protein Concentration

Contributions: 

Objective Gibson

Material/Method Gibson

Data/Result a) c) LaPlant; b) d) e) Gibson

Discussion a)-d) Gibson

Reference LaPlant

Objective

This experiment aimed to use Bio-Rad Microassay Coomassie Brilliant Blue G250 dye to measure the concentration of isolated rabbit immunoglobulin G (IgG) from DEAE (diethylethanolamine) Affi-Gel® eluate fractions from a previous experiment. Bovine serum albumin (BSA) protein concentration slope equation was obtained with the same dye at the same time as the IgG reaction with Coomassie Brilliant Blue G250 dye. IgG quantities for all fractions were calculated based on the BSA protein quantity slope equation. Finally, IgG concentrations for all fractions were calculated and plotted, then compared to the previous experiment’s light absorbing at 280 nm wavelength (A280) measurements of eluates.

Material And Methods (1)

Isolating IgG with Ammonium Sulfate Precipitation and Chromatograph

Two weeks before Bio-Rad Microassay, 1.1 mL of whole rabbit serum was precipitated by 44% and then 40% of saturation concentration of ammonium sulfate solution in an ice bath in a previous experiment. After centrifugation, the pellet was redissolved in 1.1 mL of degassed 0.02 M Tris solution with hydrochloric acid (Tris-HCl), pH 8.0, and dialyzed with a 12-14 kDa molecular weight cut-off (MWCO) membrane for one week.

One week before Bio-Rad Microassay, a DEAE Affi-Gel® chromatography column with a 36 mL/hour flow rate of Tris-HCL, pH 8, was used to produce 21 mL of eluates with previous week’s dialyzed suspension into 21 test tubes, numbered 0 through 21. Tube 0 was discarded. Tube 1, 2, 5, 11, and 20’s content were used to plot UV light absorbance at 280 nm wavelength (A280) for IgG. The contents of test tubes 2, 5, and 11 were (due to pipetting difficulties) transferred and stored in 3 1.5 mL Eppendorf tubes while tubes 1, 3, 8, and 20 were stored in their respective eluate test tubes for one week at 4° C.

Determination of IgG Concentration by Coomassie Brilliant Blue Dye Reaction

Bovine serum albumin (BSA) at 1 mg/mL concentration in Tris solution was used as stock to load 2, 4, 6, 8, 10, and 18 µg of BSA into glass tubes with preloaded Tris-HCL (pH 7.4) to make 800 µL reagent volumes per loading map (Table 1). 100 µL of each of the previous week’s chromatography eluate tubes 1, 2, 3, 5, 8, 11, and 20, were also loaded per the loading map to make 800 µL reagent volumes. All components of all reagents were vortexed before use. Then 200 µL Coomassie Brilliant Blue dye solutions were added to each of 15 test tubes in quick successions to start the dye reaction. The tubes after adding the dye were photographed before the 5-minute reaction time started.

Table 1. Test Tubes Loading Map (1)

Tubes	Sample	Sample volume	Tris-HCl	Bio-Rad dye	595 nm average value
1	Blank	-	800 µl	200 µl
2	BSA 2 µg	2 µl BSA Stock	798 µl	200 µl
3	BSA 4 µg	4 µl BSA Stock	796 µl	200 µl
4	BSA 6 µg	6 µl BSA Stock	794µl	200 µl
5	BSA 8 µg	8 µl BSA Stock	792 µl	200 µl
6	BSA 10 µg	10 µl BSA Stock	790 µl	200 µl
7	BSA 18 µg	18 µl BSA Stock	782 µl	200 µl
8	Blank	-	800 µl	200 µl
9	Fraction#1	100 µl	700 µl	200 µl
10	Fraction#2	100 µl	700 µl	200 µl
11	Fraction#3	100 µl	700 µl	200 µl
12	Fraction#5	100 µl	700 µl	200 µl
13	Fraction#8	100 µl	700 µl	200 µl
14	Fraction#11	100 µl	700 µl	200 µl
15	Fraction#20	100 µl	700 µl	200 µl

Legend: BSA, bovine serum albumin; Stock, 1 mg/mL BSA; Bio-Rad dye, Coomassie Brilliant blue G250 dye

After 5 minutes in reaction, 100 µL of each test tube’s contents were loaded in triplicates into a 96-well MicroELISA well array per the loading map (Table 2). Each tube was vortexed immediately before plating. The 595 nm light wavelength absorbances were measured with SpectraMax spectrophotometer. 

Table 2: Loading Map for 96-well Plate (1)

Legend: Blank, Tris-HCL only reagent substrate

Data And Results (2)

Figure 1 shows the photograph of the 15 test tubes roughly 2 minutes after Coomassie Brilliant G250 dye was added.

Figure 1. BSA Protein Standard and IgG Eluate Fractions from Chromatography (2)

Caption: Upper marks 1 through 15 denotes contents of tube 1 through 15 in Table 1; lower marks 1, 2, 3, 5, 8, 11, and 20 denotes chromatography Eluate Fractions in their respective tube numbers. 

Tube 1 contained a blank Tris-HCl solution with the experiment dye and no brilliant blue coloration during the reaction. Tube 2 through 7 contained 2, 4, 6, 8, 10, 18 µg BSA protein and had progressively darker blue coloration. Tube 8 also contained a blank Tris-HCl solution with the experiment dye and no blue coloration. Tube 9 through tube 15 (fraction 1, 2, 3, 5, 8, 11, and 20) had increasing coloration and then decreasing coloration. 

The samples were then loaded into a 96-well transparent plate in accordance with the loading map in Table 2.

Figure 2. Sample loading of 96-well Transparent plate (7)

Caption: The samples were loading in accordance to the loading map(Table 2)

Figure 3 shows the photograph of SpectraMax spectrophotometer reading result of MicroELISA array A595 (light absorbance at 595 nm wavelength) after 5 minutes of reaction time.

Figure 3. SpectraMax Readings Of 96-Well MicroELISA Array (2)

Table 3 shows the standard BSA A595 reading with triplicate average values and calculated protein concentrations; table 4 shows the experiment chromatography eluates fractions’ A595 reading with triplicate average values and calculated protein concentrations. 

Table 3. A595 Readings And Averages Of Bio-Rad Microassay With BSA Standards (2)

Tube #	Sample ID	Stock / Sample volume (µl)	Tris-HCl (µl)	Bio-Rad dye (µl)	Absorbance @ 595 nm (OD)	Average absorbance (OD)	Corrected absorbance (OD)	Concentration (µg/mL)
1	Blank	-	800	200	0.100	0.097	-	-
					0.100
					0.103
8			800	200	0.093
					0.093
					0.094
2	Standard 1	2	798	200	0.191	0.191	0.094	2
					0.191
					0.191
3	Standard 2	4	796	200	0.214	0.215	0.118	4
					0.214
					0.217
4	Standard 3	6	794	200	0.267	0.262	0.165	6
					0.262
					0.258
5	Standard 4	8	792	200	0.256	0.250	0.153	8
					0.244
					0.25
6	Standard 5	10	790	200	0.245	0.243	0.146	10
					0.243
					0.242
7	Standard 6	18	782	200	0.276	0.278	0.181	18
					0.282
					0.277

Legend: Red text denotes outlier data points that are disregarded; Standard refers to BSA solutions with a known protein quantity as concentration reference points; OD optical density, measured by light absorbance at wavelength 595 nm.

Table 4. A595 Of Bio-Rad Microassay with Experiment Chromatography Eluates (2)

Tube #	Sample ID	Stock / Sample volume (µl)	Tris-HCl (µl)	Bio-Rad dye (µl)	Absorbance @ 595 nm (OD)	Average absorbance (OD)	Corrected absorbance (OD)	Concentration (µg/mL)
9	Fraction #1	100	700	200	0.083	0.086	-0.011	-209
					0.088
					0.087
10	Fraction #2	100	700	200	0.118	0.119	0.022	-144
					0.118
					0.121
11	Fraction # 3 50% ascending	100	700	200	0.118	0.118	0.021	-146
					0.115
					0.121
12	Fraction #5  Peak	100	700	200	0.114	0.114	0.017	-154
					0.111
					0.117
13	Fraction # 8 50% descending	100	700	200	0.136	0.138	0.041	-107
					0.137
					0.141
14	Fraction #11	100	700	200	0.113	0.115	0.018	-152
					0.116
					0.115
15	Fraction #20	100	700	200	0.099	0.103	0.006	-175
					0.1
					0.11

Legend: Fraction Peak, chromatography experiment’s eluate with highest A280; Fraction 50% ascending, chromatography experiment’s eluate before the output fluid reached the highest A280; Fraction 50% descending, chromatography experiment’s eluate after the output fluid reached the highest A280 and diminished A280 reached half of the peak value; OD optical density, measured by light absorbance at wavelength 595 nm.

Figure 4 shows the standard BSA protein quantity A595 slope with its trend line equation and R-square value. The data points are from Table 3 absorbance table, outlier sample 3 (6 µl) excluded.

Figure 4. BSA Standard Curve After An Outlier Was Excluded (2)

Caption: X-axis was the concentration of BSA solution added to a well at a 100 µL volume

Table 5 shows the calculated IgG concentration of the experiment chromatography output. The calculations are based on linear statistical interpolations.

Table 5. IgG Eluate Fractions Concentrations (2)

Tube #	Corrected avg absorbance (y)	Protein  µg in 1 mL (x)	Dilution Factor	Concentration of Original Column Fraction(µg/mL)
9	-0.011	-20.91	1:10	-209.2
10	0.022	-14.44	1:10	-144.4
11	0.021	-14.64	1:10	-146.4
12	0.017	-15.42	1:10	-154.2
13	0.041	-10.72	1:10	-107.2
14	0.018	-15.29	1:10	-152.9
15	0.006	-17.58	1:10	-175.8

Legend: Corrected avg absorbance, this is the average of triplicates for a test tube fraction subtracted by blank background Tris-HCL solution absorbance.

The statistical interpolation line equation was derived as follows,

y = 0.0051x+0.0955 ,

y – 0.0955 = 0.0051x ,

x = (y – 0.0955)/0.0.0051 .  

And the dilution factor of each fraction made into the 1 mL reagents solution was calculated as 

100 µL eluate / (100 µL eluate + 200 µL Coomassie Brilliant Blue dye + 700 µL Tris-HCL) = 100 µL / 1000 µL = 1/10 .

Sample calculation for tube 13 concentration of original column fraction follows: tube 13 corrected average absorbance y value was 0.041, and putting 0.041 in the interpolation equation, x = (0.041– 0.0955)/0.0051 = -10.719 (µg in 1 mL). Multiplying 10 for the dilution factor 1:10 gave the concentration of the original column fraction, -10.719 µg/mL × 1000 µL/mL = -107.2 µg/mL.

In Figure 5, the orange curve shows A595 of chromatography output fractions with varying IgG concentrations reaction with Bio-Rad Microassay Coomassie Brilliant Blue G250 dye. The blue curve shows the A280 of chromatography output fractions with varying IgG concentrations one week after the start of the two-week experiment protocol when column chromatography produced the eluate fractions. 

Figure 5. A595 Of Chromatography Output Fractions With Varying IgG Concentrations (2)

Caption: ASP, ammonium phosphate precipitation

In Figure 6, the orange curve shows the same A595 of chromatography output fractions as Figure 5, excluding fractions 2, 5, and 11, which were stored in Eppendorf centrifugation tubes for one week before Bio-Rad Microassay. The blue curve shows the A280 of chromatography output fractions with varying IgG concentrations, the same as in Figure 5.

Figure 6. A595 Of Chromatography Output Fractions Excluding Fractions Stored in Eppendorf Tubes (2)

Discussion

Bio-Rad assay is a chromogenic reaction, modified from Bradford’s original assay procedure in 1976, to estimate the protein concentration of a given soluble protein solution. It uses Coomassie Brilliant Blue G250 dye to bind to proteins in solution, and the binding of a dye molecule to a protein molecule changes the dye’s color, shifting the absorbance light wavelength from 465 nm to 595 nm (3). The accurate linear range for standard Bio-Rad is 0.2 mg/mL to 1.5 mg/mL of proteins according to manufacturer documentation (3).  Bio-Rad is frequently used as a protein concentration assay due to its quick reaction time and stable color absorption after reaction, according to the lab manual (1). The absorbance trend line with known bovine serum albumin (BSA) quantities is used as a stand concentration equation to calculate an unknown protein solution’s concentration with statistical interpolation. To establish the standard concentration equation, test tubes with BSA concentrations between 0.2 mg/mL and 1.5 mg/mL are made to react with the assay dye. Simultaneously, unknown concentration protein solutions must be set up with assay dye reaction. Both known and unknown concentration solutions' light absorbance at 595 nm is measured after 5 minutes of reaction time. A variant of Bio-Rad assay, Bio-Rad Microassay has a higher sensitivity protein. The difference between standard Bio-Rad assay and Bio-Rad Microassay is 1) the different accurate linear ranges of protein concentration and 2) the concentration ratio used between the assay dye and the protein in solution. Bio-Rad Microassay has an accurate linear range of protein concentration between 1.2 µg/mL to 25 µg/mL, more sensitive than standard Bio-Rad assay. Standard Bio-Rad assay uses 5 mL diluted dye with 100 µL protein solution, while Bio-Rad Microassay uses 200 µL concentrated dye with 800 µL diluted protein solution (3).

In this experiment, Bio-Rad Microassay was used for its sensitivity for the rabbit test serum immunoglobulin concentrations between 1.2 µg/mL to 25 µg/mL. According to literature, the concentration of IgG in rabbit serum is about 70 µg/mL, which is lower than standard Bio-Rad assay can linearly accurately interpolate (4).   

The standard, known BSA concentration-versus-A595 equation, y = 0.0051x+0.0955, was obtained in Figure 4 with a  R-square value of 0.899. This relatively high R-square meant that interpolations of unknown protein concentrations could be obtained with some confidence.

Table 5’s calculation result shows that tube 13 (fraction 8) had the highest (least negative) concentration of proteins; tubes 9 and 15 (fractions 1 and 20) had the lowest (most negative). Table 5 differs from the expected result in which tube 12 (fraction 5) was expected to have the highest concentration since it had the highest IgG UV light absorbance at 280 nm. The interpolation equation had a negative x-axis intercept, meaning that concentrations lower than the known BSA concentrations could be calculated as having negative concentration, as shown in Table 5’s “Protein µg in 100 µL” and “Original Column Fraction(µg/mL)” columns. This also meant that the test subject’s protein concentration was too low for accurate calculations since the negative x-axis values should ideally be avoided in assays for concentrations.

As shown in Figure 5, both the Bio-Rad protein A595 curve and IgG A280 UV absorbance were very small at the first and 20th fractions of chromatography output. Both A595 and A280 increased in the middle fractions of the elution. This meant that IgG was a protein component of the eluates and that the concentration of IgG was roughly proportional to the overall protein concentration, similar to the lab manual descriptions (1).

The purpose of Bi-Rad Microassay protein concentration plotting by A595 in conjunction with A280 UV absorbance of IgG eluate curve comparison in this experiment was to compare the elution speed of IgG to the overall protein content elution speed, which would infer the protein species identity relation with IgG. The lab manual descriptions align the A280 and A595 absorbance curves when superimposed, implicating that the overall protein product should be largely IgG (1).

However, Figure 5’s A595 curve dipped at the peak fraction (fraction tube 5) of A280 UV absorbance. This differed from the lab manual descriptions, as the high A280 UV absorbance should indicate a high protein concentration fraction unless there were contaminant protein species that did not elute at the same speed as IgG or had other experiment errors. According to the lab manual, the proteins should elute out of DEAE Affi-Gel volume at nearly the same speed to produce roughly identical A595 curve and A280 curve. The fractions 2, 5, and 11 eluates were stored in Eppendorf centrifugation tubes, which were different from the other fractions’ storage tubes and could be a source of experiment variables.  

The overall 2-week protocol first produced ammonium phosphate precipitation of a broad spectrum of protein species in rabbit serum but diluted and removed proteins smaller than 12-14 kDa. Dialysis and DEAE Affi-Gel Blue Gel column chromatography were completed in the middle of the 2-week protocol. The Ouchterlony double diffusion tests verified the removal of many protein species by the contrast of double diffusion identity arc between commercially prepared rabbit IgG and peak A280 eluate of chromatography in Ouchterlony wells to a thick halo around the post-ammonium phosphate precipitation starting material (2). The peak A280 eluate was located in fraction 5 of the chromatography output. At the end of the 2-week protocol, rabbit IgG was assayed with Bio-Rad Microassay, showing the highest protein concentration in the eluate fractions between 6 and 8 (Figure 5, Figure 6). The experiment result was unexpected from the lab manual descriptions, where Bio-Rad assay peak protein concentration was at the same fraction as peak A280 fraction during chromatography (1). 

Storing IgG protein in Eppendorf centrifugation tubes for fractions 2, 5, and 11 for 1 week was possibly a cause of sequestering protein IgG, according to literature (5, 6). The Eppendorf tubes in this experiment were made of polypropylene (5), and protein nonspecific bindings to polypropylene had been reported before by researchers (6). The binding and sequestering of IgG protein onto Eppendorf tube walls might have reduced the IgG protein concentration in tubes 2, 5, and 11, contributing to the dip of the A595 curve at the tube 5 fraction of Figure 5. When data points of samples stored in Eppendorf were disregarded, as shown in Figure 6, the A595 protein absorbance rose rapidly from fraction 1, reaching the absorbance peak between fraction 6 and fraction 8, and gradually subsided, similar to the outline of the A280 absorbance curve.

In conclusion, this experiment produced protein concentration estimates in rough agreement with the A280 absorbance curve. Further studies can improve the protein concentration calculation from the standard BSA interpolation slope by increasing the overall test solutions’ concentration.

References 

1. Royan, S.V. & A. Alton. (2024). Immunology Laboratory Manual Fall 2024. [Unpublished Immunology Laboratory Manual]. Department of Biological Sciences, University of Massachusetts, Lowell.

2. Gibson, John. (2024). Immunology Laboratory Notebook Fall 2024. [Unpublished Immunology Laboratory Notebook]. Department of Biological Sciences, University of Massachusetts, Lowell.

3. Bio-Rad Laboratories. (n.d.). Protein quantification using the Bradford method. Bio-Rad Laboratories. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT33.pdf

4. Remberger, Mats, and Berit Sundberg. “Low serum levels of total rabbit-IgG is associated with acute graft-versus-host disease after unrelated donor hematopoietic stem cell transplantation: results from a prospective study.” Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation vol. 15,8 (2009): 996-9. doi:10.1016/j.bbmt.2009.04.013

5. Suttirojpattana, Tayita et al. “Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development.” Acta veterinaria Hungarica vol. 65,4 (2017): 546-555. doi:10.1556/004.2017.053

6. Redcenko, Oleksij et al. “Simplified PCR-Based Quantification of Proteins with DNA Aptamers and Methylcellulose as a Blocking Agent.” International journal of molecular sciences vol. 25,1 347. 26 Dec. 2023, doi:10.3390/ijms25010347

7. LaPlant, K. (2024b). 96-Well Loading  [Photograph]. Department of Biological Sciences, University of Massachusetts, Lowell.