Protein identification analysis (In-gel)
In order to understand the protein composition of a sample, we can use mass spectrometry, which can analyze large scale of samples, and the results are more direct than using indirect detecting methods like antibodies, which can be affected by the specificity of the antibody.
We can accept in-gel or in-solution samples from customers, which is convenient for customers.
In-gel samples are suitable for customers that require analyzing proteins from 1-D or 2-D gel bands. In-solution samples are suitable for customers that require analyzing proteins from more complexed samples like cell, plasma, and tissue. Of course, the efficiency of analyzing gel samples are better than in-gel samples.
We can accept in-gel or in-solution samples from customers, which is convenient for customers.
In-gel samples are suitable for customers that require analyzing proteins from 1-D or 2-D gel bands. In-solution samples are suitable for customers that require analyzing proteins from more complexed samples like cell, plasma, and tissue. Of course, the efficiency of analyzing gel samples are better than in-gel samples.
The proteins in the sample will be digested into small peptides by trypsin, and then use LC/MS/MS machine (1DLC, LTQ-Orbitrap MS) to separate hydrophobicity differences of the peptides. After that, the peptides will be further separated according to m/z ratio. We will pick the peptides of interest and break down the peptides even more. The peptide will be broken down into y- or b- ions, and after calculating mass difference between the nearby y- or b- ion, we can obtain the amino acid composition of the peptide (see the picture below).
Service Specification
| In-gel | Basic Analysis | Standard Analysis | Advance Analysis |
| Specification | 1D, 70 min | 1D, 70 min * 5 regions | 1D, 70 min * 15 regions |
| Protein number can be quantified | 100-500 | 100-1800 | 500-3000 |
Note: For standard and advance analysis, please provide the sample as solution form.
Working day not including delivery time: 2 weeks
1. In-gel protein identification: Basic analysis
(a) Sample type: Gel slice (please provide gel image with staining)
(b) Gel size: ≤3 mm x 8 mm x 1.5 mm (Height x Width x Thickness)
(c) Platform: 1DLC, LTQ-Orbitrap MS
2. In-gel protein identification: Standard/Advance analysis
(a) Sample type: Cell pellet, Protein extracts
(b) Sample volume: ≤50 uL
(c) Sample concentration: ≥0.2 ug/uL (silver stain), ≥1 ug/uL (Coomassie blue stain)
(d) Platform: Hoefer SE260, 1DLC, LTQ-Orbitrap MS
Please click the link below for the specification of Proteomics service
Sample requirement
1. Summary report. pdf
2. Protein-peptide report. xlsx
Q1. Can you analyze every animal or species?
A: Please provide us the species of your sample, we will use the database of NCBI or Swiss-Prot to analyze to see if you sample is suitable for us to analyze
Q2. If the protein of interest in the sample is relatively low amount, will there be a chance that we will not be able to identify the protein?
A: Please provide us more than 20 ug of the protein of interest and we will do gel electrophoresis to assess whether or not there will be a chance the protein will not be identified. This normally happens in the in-solution samples with using 1 fraction. To solve this matter we also provide more than 6 fractions for protein identification, or you can alternatively use in-gel method to decrease the chance of unidentify your protein of interest
Q3. What should I pay attention to when preparing my sample?
A: You should avoid getting your samples contaminated. For example, you will identify a lot of keratin if you sample is contaminated during protein extraction. You should also avoid collecting the serum and growth factors in the medium when collecting cells. For blood samples, we need to have an additional charge for abundant protein removal, or else you will only identify a lot of albumins.
Q4. If there is no protein signal after identification in my sample, how will you charge this case?
A: If you have done SDS-PAGE to confirm your target protein before you give us the sample, this case will be free of charge, or we will change the method by using in-gel method to collect the specific target protein band to identify.
If you cannot provide us the SDS-PAGE or provide enough sample to do in-gel method to confirm, we will assume this is a problem with the sample and would still charge you for the service.
Q5. Can the protein in the organic phase of TRIZOL RNA extraction be used for protein identification?
A: Yes, we suggest in-gel digestion method. For further details, please contact our technician.
Q6. Can you identify peptides? Or can you identify digested samples?
A: No. Most of the peptides have been digested with unkown enzyme, which will effect the analyzation of the results.
Q7. Can you do this service with blood samples?
A: If you do protein identification, we suggest you combine with abundant protein removal service to exclude the high abundant proteins (e.g. albumin). If you don't the results will be affected greatly. For protein quantification, we suggest label-free quantification. For further details, please contact our technician.
Q8. Is cytokine-related proteins be identified using LC-MS/MS system?
A: If you wish to detect the cytokine in cell supernatant, we do not suggest you use LC-MS/MS platform. For further details please contact our technician.
Q9. Are there any limitations for in-gel samples?
A: The size of in-gel samples should not exceed 3x8x1.5mm. We will charge additionally if the gel size is bigger than our standard. For sequencing analysis, we don't suggest in-gel samples. In-gel samples will effect the efficiency of peptide elution, which leads to lower coverage.
A: Please provide us the species of your sample, we will use the database of NCBI or Swiss-Prot to analyze to see if you sample is suitable for us to analyze
Q2. If the protein of interest in the sample is relatively low amount, will there be a chance that we will not be able to identify the protein?
A: Please provide us more than 20 ug of the protein of interest and we will do gel electrophoresis to assess whether or not there will be a chance the protein will not be identified. This normally happens in the in-solution samples with using 1 fraction. To solve this matter we also provide more than 6 fractions for protein identification, or you can alternatively use in-gel method to decrease the chance of unidentify your protein of interest
Q3. What should I pay attention to when preparing my sample?
A: You should avoid getting your samples contaminated. For example, you will identify a lot of keratin if you sample is contaminated during protein extraction. You should also avoid collecting the serum and growth factors in the medium when collecting cells. For blood samples, we need to have an additional charge for abundant protein removal, or else you will only identify a lot of albumins.
Q4. If there is no protein signal after identification in my sample, how will you charge this case?
A: If you have done SDS-PAGE to confirm your target protein before you give us the sample, this case will be free of charge, or we will change the method by using in-gel method to collect the specific target protein band to identify.
If you cannot provide us the SDS-PAGE or provide enough sample to do in-gel method to confirm, we will assume this is a problem with the sample and would still charge you for the service.
Q5. Can the protein in the organic phase of TRIZOL RNA extraction be used for protein identification?
A: Yes, we suggest in-gel digestion method. For further details, please contact our technician.
Q6. Can you identify peptides? Or can you identify digested samples?
A: No. Most of the peptides have been digested with unkown enzyme, which will effect the analyzation of the results.
Q7. Can you do this service with blood samples?
A: If you do protein identification, we suggest you combine with abundant protein removal service to exclude the high abundant proteins (e.g. albumin). If you don't the results will be affected greatly. For protein quantification, we suggest label-free quantification. For further details, please contact our technician.
Q8. Is cytokine-related proteins be identified using LC-MS/MS system?
A: If you wish to detect the cytokine in cell supernatant, we do not suggest you use LC-MS/MS platform. For further details please contact our technician.
Q9. Are there any limitations for in-gel samples?
A: The size of in-gel samples should not exceed 3x8x1.5mm. We will charge additionally if the gel size is bigger than our standard. For sequencing analysis, we don't suggest in-gel samples. In-gel samples will effect the efficiency of peptide elution, which leads to lower coverage.