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:

  1. Compare batch-to-batch HCP profile
  2. 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 could 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

HCP Profiling Analysis: 2D image of CHO_S1

CHO_S2: 1979 Spots

HCP Profiling Analysis: 2D image of CHO_S2

CHO_S3: 2378 Spots

HCP Profiling Analysis: 2D image of CHO_S3

CHO_S1 / CHO_S2

HCP Profiling Analysis: 2D overlay image of CHO_S1 vs CHO_S2

CHO_S1 / CHO_S3

HCP Profiling Analysis: 2D overlay image of CHO_S1 vs CHO_S3

CHO_S2 / CHO_S3

HCP Profiling Analysis: 2D overlay image of CHO_S2 vs CHO_S3

B. HCP Quantitative analysis

The following quantification analysis can be performed to assess and compare the global protein profiling of HCP samples:

  1. Spot counting of each sample
  2. Spot overlapping between samples: % spots overlapped between HCP samples
  3. Protein level comparison (protein ratio) between samples: Using our standard or customer set cutoffs, % spots increased, decreased or similarly expressed between HCP samples.
  4. 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:

GelS1 spotsS2 spotsOverlapping spots% S1 spots
covered by S2
% S2 spots
covered by S1
124451979188877.2%95.4%
GelS1 spotsS3 spotsOverlapping spots% S1 spots
covered by S3
% S3 spots
covered by S1
124452378218589.4%91.9%
GelS2 spotsS3 spotsOverlapping spots% S2 spots
covered by S3
% S3 spots
covered by S2
119792378183692.8%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.

GelComparisonProtein Ratio: 2 Fold CutoffProtein Ratio: 5 Fold Cutoff
Decreased %Similar %Increased %Decreased %Similar %Increased %
1CHO_S2 / CHO_S126.3%54.6%19.1%8.3%89.7%2.0%
CHO_S3 / CHO_S116.9%67.7%15.4%2.6%95.6%1.8%
CHO_S3 / CHO_S220.2%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

HCP image of CHO_S2 vs CHO_S1

CHO_S3 / CHO_S1

HCP image of CHO_S3 vs CHO_S1

CHO_S3 / CHO_S1

HCP image of CHO_S3 vs CHO_S2


B4. HCP Quantitation:

#Host Cell ProteinCHO_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

HCP image of CHO_S1 vs S2 vs 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 standard sample preparation is covered by the DIGE gel cost. Such samples should have protein concentration in the range of 5-20 mg/ml. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, 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 DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
2D DIGE Analytical Gel (2 CyDye labeling)101A2$1225101N2$1500
2D DIGE Analytical Gel (3 CyDye labeling)101A3$1375101N3$1685
HCP Quantitative Analysis202A-HCPcall for price202N-HCPcall for price

Price covers:

  • Experimental design
  • Standard sample preparation and protein concentration determination
  • Fluorescent dye labeling using CyDye
  • IEF and SDS-PAGE
  • Gel image scan using Typhoon scanner
  • Image analysis (ImageQuant)
  • Data Report