Immunoprecipitation (IP) Proteomics
IP proteomics, which identifies interacting proteins from Co-IP experiments, can be challenging due to the low abundance of pull down proteins in the presence of high abundant antigen and/or antibody proteins. Our 2D DIGE & Mass Spectrometry platforms offer unique advantages in:
- High resolution: Well-resolved pull-down (target) proteins in the presence of abundant proteins
- High specificity: Filter out un-specific binding by including control sample
- Low cost: 3 samples in 1 gel; only ID the target spots
- Fast turnaround: 5-7 days
For gel layout, please view the study designs based on the total number of samples.
We also offer nanoLC-MS/MS to identify proteins in IP complex. Please note that if IP was performed with non-cross linking antibody (example 1), 2D DIGE is a much better choice, as nanoLC-MS/MS detection of IP products would be highly compromised.
Example 1: IP using non-cross linking antibody
When performing IP using non-cross linking antibody, the products contain high-abundance antibody proteins. The image here shows 2 IPs using K8/18 antibody at different phases of cell cycle (G0/G1 and G2/M). The objective of this project is to identify cell cycle-dependent K8/18 associated proteins. The circles spots in yellow, red and green correspond to similarly, increased and decreased proteins, respectively, between the 2 phases. All circled spots are clearly resolved in the presence of high levels of IgGs.
IP from G0/G1 / IP from G2/M
Example 2: IP using cross linking antibody.
When performing IP using cross linking antibody, the products are much more cleaner without antibody proteins. The image here shows 2 IPs using cross-linking antibody against 2 different HT29 cell lysates (Control and Treated). The objective of this project is to identify different associated proteins due to treatment. The circles spots in yellow, red and green correspond to similarly, increased and decreased proteins, respectively, between the 2 IP samples. All circled spots are clearly resolved.
IP from Control / IP from PV-treated
Please follow these general principles regarding biohazardous materials.
Sample amount: 50-100 µg of IP proteins from each sample. This will be sufficient to cover the entire 2D DIGE procedure including preparative gel, spot picking and protein ID.
You may send IP beads or samples in elution buffer that you would normally use. If you send IP beads, please do the IP and washing procedure in the same way you would normally do. At the end, please transfer the beads to a 1.5 ml Eppendorf tube, wash the beads once with 0.5 ml of HPLC-grade water. Please remove the water as completely as possible. The IP bead pellet can be kept at -80oC and sent to us on dry ice. We will elute protein complex from the beads using appropriate buffers.
|Services Description||Academic/Governmental Labs||Industrial/Commercial Labs|
|IP Proteomics: Customized Sample Preparation 1||600A-DIGE||TBD||600N-DIGE||TBD|
|2D DIGE Preparative Gel (1 CyDye labeling)* 2||401A||$800||401N||$960|
|2D DIGE Preparative Gel (2 CyDye labeling)* 2||102A2||$1200||102N2||$1500|
|2D DIGE Preparative Gel (3 CyDye labeling)* 2||102A3||$1295||102N3||$1595|
|Spot Picking per Gel* 3||103||$250 per 96 spots||103||$250 per 96 spots|
|Protein ID by Mass Spectrometry (MALDI-TOF/TOF) 4||104A||$125 per spot||104N||$140 per spot|
|Pathways Analysis After Protein ID 5||203A||call for a quote||203N||call for a quote|
* Prices are discounted and only apply for customers who perform protein identification at Applied Biomics
1. Price covers:
- Elute IP proteins from beads (If applicable)
- Concentrate eluted proteins
- Clean up sample
- Exchange into 2D DIGE buffer
- Measure protein concentration
2. Price covers:
- Experimental design
- Sample preparation and protein concentration determination
- Fluorescence dye labeling using CyDye (1 sample for 1 CyDye labeling; 2 samples for 2 CyDye labeling; 3 samples for 3 CyDye labeling)
- IEF and SDS-PAGE
- Gel image scan using Typhoon scanner
- Spot picking design
3. Price covers:
- Set up spot picking
- Pick spots
- Free storage at -80C for 6 months.
2-4. Turnaround time starting from Preparative gel to sending protein ID report is 7-9 business days. We offer high sensitivity protein identification from gel spots or bands using the latest technologies in mass spectrometry. In each run, four sensitivity standards in the amount of 1-10 femtomole are included. Our sensitivity is the range of 1-2 femtomole. Please view protein ID procedure for detailed steps of this service. Price covers the following:
- Gel treatment
- In-gel trypsin digestion
- Peptide extraction
- Database search against NCBI database using MASCOT
- Data report (example below)
5. Pathway Analysis Service is provided only for customers doing the 2D DIGE and Protein ID services with Applied Biomics. The report will include the major pathway list with the following information:
- Functional groups
- Genes in each functional group
- Enrichment score
- Statistic p-value and FDR