HCP Profiling Analysis: 2D
Our HCP Profiling analyses detect, quantify and compare HCP compositions of 3 HCP samples in 1 test. For projects with more than 3 samples, Cross-gel analysis will be performed using customers pre-set control. This platform has been successfully applied in the following areas:
- Compare batch-to-batch HCP profile
- Track purification process
The analysis contains two components:
- HCP Image Analysis: Direct visualization and comparison of HCP profile such as compositions, modifications and fragmentations that no other platform can achieve.
- HCP Quantitative Analysis: Total spot count of each sample, spot overlapping between samples, protein ratios between samples, individual HCP quantity, total HCP quantity and DS quantity.
A. HCP Image analysis
Gel images of each sample and overlay of two sample images will be generated. In the overlay images, spots with green, red and yellow color indicate decreased, increased and similar expression, respectively.
Example: Image analysis of HCP samples CHO_S1, CHO_S2 and CHO_S3
CHO_S1: 2445 Spots
CHO_S2: 1979 Spots
CHO_S3: 2378 Spots
CHO_S1 / CHO_S2
CHO_S1 / CHO_S3
CHO_S2 / CHO_S3
B. HCP Quantitative analysis
The following quantification analysis can be performed to assess and compare the global protein profiling of HCP samples:
- Spot counting of each sample
- Spot overlapping between samples: % spots overlapped between HCP samples
- Protein level comparison (protein ratio) between samples: Using our standard or customer set cutoffs, % spots increased, decreased or similarly expressed between HCP samples.
- HCP and DS ppm: Quantification of specific HCP and DS spots
Example: Quantification analysis of HCP samples CHO_S1, CHO_S2 and CHO_S3
B1. Spot Counting of each sample: Number of detected spots for each sample
- CHO_S1: 2445 Spots
- CHO_S2: 1979 Spots
- CHO_S3: 2378 Spots
B2. Spot Overlapping between samples:
| Gel | S1 spots | S2 spots | Overlapping spots | % S1 spots covered by S2 | % S2 spots covered by S1 |
|---|---|---|---|---|---|
| 1 | 2445 | 1979 | 1888 | 77.2% | 95.4% |
| Gel | S1 spots | S3 spots | Overlapping spots | % S1 spots covered by S3 | % S3 spots covered by S1 |
| 1 | 2445 | 2378 | 2185 | 89.4% | 91.9% |
| Gel | S2 spots | S3 spots | Overlapping spots | % S2 spots covered by S3 | % S3 spots covered by S2 |
| 1 | 1979 | 2378 | 1836 | 92.8% | 77.2% |
B3. Protein ratios between samples: The table below shows the % spot numbers in each category using 2-fold and 5-fold of protein ratios as a cutoff.
| Gel | Comparison | Protein Ratio: 2 Fold Cutoff | Protein Ratio: 5 Fold Cutoff | ||||
|---|---|---|---|---|---|---|---|
| Decreased % | Similar % | Increased % | Decreased % | Similar % | Increased % | ||
| 1 | CHO_S2 / CHO_S1 | 26.3% | 54.6% | 19.1% | 8.3% | 89.7% | 2.0% |
| CHO_S3 / CHO_S1 | 16.9% | 67.7% | 15.4% | 2.6% | 95.6% | 1.8% | |
| CHO_S3 / CHO_S2 | 20.2% | 52.1% | 27.8% | 3.0% | 87.3% | 9.8% | |
Below is spot map showing spot distribution with 2-fold cutoff: Red, green and yellow indicate spots with increased>2-fold, decreased>2-fold and within 2-fold, respectively.
CHO_S2 / CHO_S1
CHO_S3 / CHO_S1
CHO_S3 / CHO_S1
B4. HCP Quantitation:
| # | Host Cell Protein | CHO_S1 (ppm) | CHO_S2 (ppm) | CHO_S3 (ppm) |
|---|---|---|---|---|
| 01 02 03 04 05 06 07 08 09 10 11 12 | Endoplasmin Alpha_actinin_4 Hsp72 PDIA1 Hsp60 G3P EF1B 14-3-3 PEBP1 Calmodulin_2 | 1994 201 6306 6723 713 5620 2305 3010 3405 454 2727 2456 | 137 152 7163 6964 38 5689 1874 17731 4883 759 1681 656 | 2819 278 17025 6805 2541 9472 206 1546 1571 500 1650 2153 |
CHO_S1 / CHO_S2 / CHO_S3
Sample Info
Please follow these general principles regarding biohazardous materials and buffer condition in getting your samples ready.
Protein amount for each gel: 20-50 µg of total protein from each sample.
Protein concentration: 5-20 mg/mL
Buffer: Please feel free to submit samples in whichever buffer that you would normally use. The sample preparation including protein extraction (if applicable), sample cleaning and measuring protein concentration is not covered by the 2D DIGE gel cost. Additional charge may apply on samples with concentration less than 4 mg/mL, or require significant amount of the extra work such as protein eluting, sample pooling, extensive buffer exchange or serum abundant protein depletion.
Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.
Pricing
| Services Description | Academic/Governmental Labs | Industrial/Commercial Labs | ||
|---|---|---|---|---|
| Code | Price | Code | Price | |
| 2D DIGE Analytical Gel (2 CyDye labeling) | 101A2 | $1259 | 101N2 | $1569 |
| 2D DIGE Analytical Gel (3 CyDye labeling) | 101A3 | $1399 | 101N3 | $1769 |
| HCP Quantitative Analysis | 202A-HCP | call for price | 202N-HCP | call for price |
Price covers:
- Experimental design
- Measure protein concentration
- Fluorescence dye labeling using CyDye (2 samples for 2 CyDye labeling; 3 samples for 3 CyDye labeling)
- 1st dimension IEF and 2nd dimension SDS-PAGE
- Gel image scan using Typhoon scanner
- Two hour data analysis covering image and DeCyder analysis
- Data report
Price does NOT cover:
- Sample preparation
- Manual matching spots between different gels
- Manual inspection of selected spots by DeCyder
- Screen shots of 3D view of each spot
- Preparative gel, spot picking, protein ID: Please note that Analytical gels do not contain sufficient amount of protein for protein identification. If you find some interesting spots you would like to move forward to perform Protein ID, we will need to run a Preparative gel. If the project contains only 1 Analytical gel, 1 Preparative gel is needed. If the project contains 2 or more Analytical gels, most likely only 1-2 Preparative gels are needed. Please view FAQ #7 for Why 2 stages: Analytical Gel and Preparative Gel.