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Automated analysis of proteins using a microfluidics system

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Authored by: Caliper Life Sciences


(Originally published as Caliper Life Sciences Application Note 100; reprinted with permission)

Contents

Introduction

Although SDS-PAGE is the traditional method for protein analysis, data can be variable and results are qualitative. Parts of the gel process can be automated, but a significant amount of manual interaction is still required. Microfluidic assays are proven methods for protein analysis in laboratories requiring more information in an expedient manner. The LabChip system performs automatic sampling from a microtiterplate followed by electrophoresis, data analysis, and reporting. Because sample loading, injection, and separation can be precisely controlled on the microfluidic chip, analytical data is highly reproducible.

Figure 1. A detailed diagram of the LabChip 90 protein chip. This is a top-down view, the sipper extends out underneath the chip.
 
Figure 1. A detailed diagram of the LabChip protein chip. This is a top-down view, the sipper extends out underneath the chip.


The Protein Express assay generates quantitative sizing, concentration, and purity data as each sample is processed. Sensitivity is comparable to mid-range colloidal coomassie stain, with a large dynamic range. The assay encompasses a wide variety of comparable gel concentrations, which allows a broader resolution range to be achieved.

Protein Assay Fundamentals

The assay is a microfluidic version of SDS-PAGE, where each step of the slab gel process - sample loading, electrophoresis, staining, destaining, and detection - is integrated into a microfluidic device. Quantitative sizing, relative concentration, and purity results are reported for each sample. Sample analysis takes approximately 35 seconds, and a full 96-well plate can be analyzed in just over an hour. Results can be viewed in three formats: a gel-like display, an electropherogram, and a tabulated results table. Detailed sample information can be imported into the software for tracking purposes. Protein data can also be exported for presentations, data archiving, or database submission.

Figure 1. A detailed diagram of the LabChip 90 protein chip. This is a top-down view, the sipper extends out underneath the chip.
 
Figure 2. Destain and detection region of the LabChip protein chip. The image on the right is an actual photo of this region



Microfluidic Chip Function

Preparation of the protein chip and samples can be completed in approximately 20 minutes. The samples are heat denatured in a high concentration of SDS. The SDS coats the protein, which results in a net negative protein surface charge that enables electrophoretic separation. The protein chip is prepared by pressure priming the microfluidic channels with gel-dye and destain solutions. The gel-dye solution serves as both a sieving matrix for the separation of the proteins and a staining solution. Once the protein chip is primed, a marker solution is pipetted onto the chip. Both the chip and the protein plate are then loaded onto the machine, and the assay is started. The protein chip performs several sequential functions as referenced in Figure 1. First, it uses vacuum applied to well 1 to aspirate approximately 170 nL of sample from the well plate through a capillary sipper and into the microfluidic channels of the chip. During this step the sample is diluted 2:1 with a marker solution, which is simultaneously drawn from well 4. This marker is subsequently used as a reference for migration time and determination of relative concentration of samples.

Next, the chip electrophoretically “loads” the marker-protein mixture into the channel between wells 3 and 8, across the separation channel. A 40 pL sample plug is then electrophoretically injected into the separation channel. A potential is applied between wells 7 and 10, which causes the individual proteins in the sample to migrate up the separation channel.

Each protein is stained with dye contained in the gel and separated into distinct bands with resolution comparable to a 4-20% SDS-PAGE gel. Protein destaining is accomplished using a dilution step achieved by electrokinetically flowing SDS-free ions into the separation channel at the destain intersection. This causes the dye-SDS-protein fluid stream to focus as shown in Figure 2. In approximately 250 milliseconds, diffusion of free SDS micelles into the SDS-free fluid results in breakup of the micelles and a significant drop in the background fluorescence. SDS micelles bound to the protein remain intact. Since the proteins are still coated with SDS-dye and retain their fluorescence, the separated protein bands are detected downstream of the dilution point by using laser induced fluorescence (LIF). Free solution dye molecules are not detected because they are only fluorescent in the hydrophobic environment of the SDS micelles.

Workflow

The assay is sufficiently flexible to accommodate a broad range of user workflows. Once the chip has been primed and loaded into the LabChip system, it can be used over an eight-hour period or until the lifetime of the chip has been reached. This translates into rapid results for both high- and low- throughput users. Laboratories that generate small sample sets can perform analysis immediately as samples become available throughout the day.

This intermittent workflow is beneficial for multiple researchers and groups, as it allows each to perform analysis with little effort. Higher-throughput laboratories generating full plates of samples can quickly analyze up to three 96-well plates. This continuous workflow provides the walk-away automation and speed of analysis required in screening environments where expediency in process decisions is key.

Materials and Methods

Hardware and Consumables:
  • Protein Express LabChip kit. The kit includes one chip and reagents for a minimum of 300 samples.
  • LabChip System with LabChip analysis software.
Sample and Chip Preparation:
  • Samples: Bio-Rad low molecular weight ladder, carbonic anhydrase from Sigma Aldrich. Phosphate buffered saline (PBS) from Dulbecco.
  • Samples were prepared in a 96-well plate (MJ Research, conical bottom plate) by mixing 2 μL of each protein sample and 7μL of denaturing buffer (assay sample buffer + 3.5% V/V b-mercap-toethanol).
  • The sample-buffer mixture was then heat denatured at 95 °C for five minutes. After cooling, 35 μL of DI water was added to each denatured sample.
  • The Protein Express chip was prepared as instructed in the Protein LabChip Kit User Guide.
Separation and Data Analysis:
  • The LabChip system and Protein Express chip separated, stained, destained and detected the proteins in each sample automatically.
  • LabChip software analyzed and reported the size, relative concentration and purity of the proteins detected in each sample.


Table 1: Protein Express Assay Specifications
Type Specification
Sizing Range Protein 100 assay:
14 - 100 kDa
Protein 200 assay:
14 - 200 kDa
Sizing Accuracy* ± 20%
Sizing CV* 10%
Resolution* ± 10% difference in
MW across the sizing
range, 50% valley
Linear Dynamic Range* 5 - 2000 ng/μL
Relative Concentration CV* 30% up to 120 kDa
relative to the ladder
Sensitivity* 5 ng/μL (10 ng) CA
Sensitivity is equivalent to
mid-range colloidal
coomassie blue stain
Analysis Time per Sample Protein 100 assay:
34 seconds/sample
Protein 200 assay:
39 seconds/sample
Chip Lifetime Three 96-well plates
Analysis Time Per Plate ~ 1¼ hr (96-well plate)
Maximum Salt Concentration 1M NaCl
* For samples in PBS


Results and Discussion

Sizing and Concentration Reproducibility

In Figure 3, six protein sample electropherograms have been superimposed to illustrate separation reproducibility. The sizing range is from 14 to 200 kDa, and this range can be easily separated in 30 seconds allowing 96 samples to be processed in approximately one hour. The first peak, designated LM, is the internal marker dye and is used for normalization of sample size and relative concentration. This automated normalization of data ensures excellent data reproducibility. Sizing and relative concentration are calculated with respect to ladder standards that are sipped at the beginning and end of each row of 12 samples.

Figure 1. A detailed diagram of the LabChip 90 protein chip. This is a top-down view, the sipper extends out underneath the chip.
 
Figure 3. Overlay of six electropherograms (identical samples) illustrates data reproducibility of the LabChip 90 system.


Specifically, a Bio-Rad low molecular weight ladder was prepared and pipetted into 96-well plates. The data in Tables 2 and 3 represent 2880 samples. The sizing accuracy and reproducibility of the assay is demonstrated in Table 2, and the relative concentration reproducibility is shown in Table 3.

Sizing and relative concentration reproducibility were tested in validation studies that probed variability across multiple users, instruments and chips.

Table 2: Protein Sizing Data
Protein Theoretical MW (kDa) Experimental MW (kDa)  % Error  % CV
Lysozyme 14.3 13.8 -3.3 1.8
Trypsin Inhibitor 21.5 21.2 -1.3 1.4
Carbonic Anhydrase 29.0 29.3 1.0 1.4
Ovalbumin 45.0 44.4 -0.1 1.3
Bovine Serum Albumin 66.7 73.7 10.4 1.5
Phophorylase B 97.4 93.9 -3.6 1.4


Table 3: Protein Concentration Data
Protein Average Concentration (μg/mL) Standard Deviation  %CV
Lysozyme 207.1 40.3 19.4
Trypsin Inhibitor 79.2 15.1 19.0
Carbonic Anhydrase 95.5 16.9 17.7
Ovalbumin 197.8 57.3 29.0
Bovine Serum Albumin 182.6 31.0 16.9
Phophorylase B 128.4 20.4 15.9


The LabChip system reports protein concentration relative to the protein ladder included in the Protein Express LabChip Kit. Quantitation accuracy is not reported in Table 2 for two reasons. First, the actual protein concentrations were unknown for these samples and second, the staining variability between different proteins (typical of most protein quantitation assays) will contribute to differences in the reported concentrations. Experiments indicate that most proteins analyzed by the LabChip system report to within 50% of their actual concentration. For more accurate concentration results, we recommend generating a standard concentration curve for the protein of interest and comparing the unknown sample to this data directly. Note that when diluting proteins to generate these curves, sample loss due to surface adsorption (to pipette tips and sample tubes) can contribute to under-reporting for the lower concentration samples. It is recommended to add 0.1% SDS to the sample stock solution before dilution to reduce the sample loss and improve quantitation accuracy.

Sensitivity and Carryover

The microfluidic assay sensitivity is comparable to colloidal coomassie stain. This is equivalent to the detection of 5 ng/μL (or 10 ng) of protein on the LabChip system. To demonstrate the range and sensitivity of the assay, carbonic anhydrase samples were prepared at concentrations between 2000 ng/μL and 2.5 ng/μL. The virtual gel image from this complete range is shown in Figure 4. The electropherogram displays the data between 25 and 2.5 ng/μL to illustrate the sensitivity. Due to the variable staining behavior of different proteins, not all proteins can be seen as low as 2.5 ng/μL.

Microfluidics 4.jpg
 
Figure 4. Samples of carbonic anhydrase were diluted to concentrations between 2000 and 2.5 ng/μL. The virtual gel data for all concentrations is shown as well as an overlay of electropherograms for the lower concentration samples.


Sample 11 shown in the virtual gel image in Figure 4 is a buffer blank run immediately after the 2000 ng/μL protein sample and demonstrates that sample carryover is less than 0.2%.

Assay Compatibility with Various Salts, Buffers and Additives

Many buffers, salts and additives have been tested to demonstrate robustness to common assay conditions. The Table 4 summarizes a subset of the reagents tested.

Table 4. Protein Express assay buffer, salt and additive compatibility. All additives were used in PBS, pH 7.5.
Buffer and Salts Additives
Tris Chloride 250 mM Octyl Glucoside 2.5%
Tris Glycine 250 mM Pluronic F68 0.1%
HEPES 500 mM Sarcosyl 10%
PBS 8 X CHAPS 0.5%
Sodium Citrate 150 mM Tween 20 0.8%
Sodium Phosphate 250 mM Triton X-100 0.6%
Sodium Acetate 600 mM SDS 2%
Sodium Chloride 1000 mM Zwittergent 3-14 0.4%
Sodium Azide 6% PEG 3350 1%
Sodium Hydroxide 500 mM Glycerol 30%
Potassium Chloride 900 mM Urea 8 M
Ammonium Bicarbonate 1000 mM Sucrose 1 M
Magnesium Chloride 300 mM DMSO 25%
Imidazole 900 mM EDTA 100 mM
PhosphoSafe   Ethanol 50%
BugBuster 2.5 X    
BPER      
POP Culture      
Insect POP Culture      


Protein Expression Monitoring Using the Protein Express Assay

There are many applications for protein sizing and quantitation using the LabChip system. These applications can include the monitoring of protein expression and solubility, analysis of column fractions and purified proteins, and antibody QC, among others.

In protein expression, cells are modified specifically to over-express proteins of interest. These cells are then lysed, followed by an extraction and purification of soluble protein. Protein analysis is commonly performed on the whole cell lysate to determine the degree of protein expression, on the supernatant to determine expressed protein solubility, and then on the partially purified fractions to determine purity. Further processing and analysis of the insoluble cell material are often conducted to confirm expression levels. Not all expressed proteins are seen in the soluble component of the cell lysate due to precipitation. Analysis of the insoluble cell material provides further verification of protein expression levels, and is performed to potentially avoid inaccurate conclusions drawn from initial data.

Conclusion

Automation of the SDS-PAGE process allows scientists to spend valuable time on experimentation and research, rather than processing slab gels. The time-consuming and labor-intensive manual slab gel process can sometimes generate variability in data that may be unreliable over time and across experiments. Using a microfluidic assay, high-quality sizing and quantitation data is presented quickly, allowing more accurate decisions to be made much sooner in the expression process. Both high- and low-throughput laboratories can take advantage of the system’s automated analysis, as workflow flexibility permits anywhere from just a few samples to multiple plates to be analyzed throughout the day as needed. Run times of approximately one hour can result in more than a 3X increase in laboratory throughput. Compatibility with microplates makes upstream automation possible by permitting automation of the entire sample preparation and analysis process, both of which are the most common bottlenecks faced by protein laboratories today. The digital data format allows results to be compared between experiments and shared easily between multiple groups at multiple sites, and simplifies database population. Microfluidic assays can be a powerful tool for protein expression, purification, production and engineering groups requiring efficient analysis of lysates, column fractions, purified proteins, and antibodies.


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