Phosphorylation Site Identification
What are the special considerations for identifying the phosphorylation site of a protein?
There are two special considerations.
- Coverage: The higher the coverage of the target protein in mass spectrometry detection, the higher the probability of the protein being modified by a modified group and the detection and site quantification.
- Proportion of modification sites: If the proportion of modification sites is low, the probability of detecting the modified peptide is low. Generally, if the proportion is greater than 30%, the peptide modification site is highly likely to be detected.
Western Blot Analysis of Phosphorylated Proteins
If the target protein is a membrane protein or a cytoplasmic protein, what do I need to be aware of?
A: If the target protein is a membrane protein or a cytoplasmic protein, the detergent used is much milder and it is best to add NaF to inhibit the phosphorylase activity.
What are the secondary antibody requirements for Western Blot for cell signalling and phosphorylation of a factor?
A: There is no requirement for secondary antibodies, it depends on your experimental conditions, generally HRP-labelled secondary antibodies are recommended.
Can I detect phosphorylated JNK and non-phosphorylated JNK on the same membrane?
A: Yes.
I would like to ask you whether to use triple decontamination lysis or loading buffer for lysing cells?
A: Use the top sample buffer, this has several advantages, it extracts the total protein and at the same time inactivates the phosphorylase.
How can I prevent dephosphorylation from occurring when preparing a Western Blot of rat brains, where the protein is located in the nucleus?
A: You can use the same buffer used to extract the total protein to extract the nuclear protein, and you can add NaF to prevent dephosphorylation.
I need to measure two antibodies, one for the phosphorylated target protein and one for the total target protein, and I'm wondering what is the best method to STRIP? I am using glycine (pH 2.9) for 15 minutes, but it doesn't seem to work?
A: You can add mercaptoethanol (same concentration as loading buffer) at 56°C for 30mins.
Is it necessary to add NaF etc. to the sample preparation for the phosphorylated antibody assay?
A: Not necessarily, NaF is a broad-spectrum phosphorylase inhibitor and it is usually best to add it, but it can be done without it.
I need to measure the total amount of the same protein and the amount of phosphorylation, but there is too much interference between them, what should I do?
A: Place the membrane in stripping buffer (SDS 2%, Tris-Cl (pH=6.7) 62.5mM, beta-mercaptoethanol 100mM) and incubate for 30 minutes at 50°C. Wash three times with TTBS and re-add primary antibody for the other antigen.
Does the phosphorylation modification of a molecule make this molecule larger in molecular weight? What if I want to detect the phosphate of a protein from tissue?
A: For each modification, the molecular weight is increased by 80. The change in electrophoretic mobility before and after phosphorylation is measured directly by SDS-PAGE, and the electrophoretic mobility of the protein slows down after phosphorylation. However, this is related to the nature of the protein, the number of sites it is phosphorylated at, the change in molecular weight before and after, etc.
What are the differences between phosphorylated western and normal western and what are the considerations?
A: Phosphorylation of proteins is a very rapid reaction, so the process of taking samples should be rapid, the samples should be fresh, and it is also best to have freshly prepared lysate. The lysate must have a protease inhibitor and a phosphatase inhibitor in it. It also depends on what amino acid the phosphorylation site of the protein being tested is, if it is tyrosine also add 1um of sodium vanidate.
Closed specialised skimmed milk powder, which is specially processed to remove fat and inactivate protein kinase and protein phosphatase activity. Inactivation of protein kinase helps to protect the wild-type protein substrate in its non-phosphorylated state. Inactivation of protein phosphatase protects the phosphorylated protein from dephosphorylation by dephosphatase, thus preserving the phosphorylated state of the protein and providing optimal western blotting without affecting the properties of the protein substrate, making it particularly suitable for protein phosphorylation and dephosphorylation studies. It is additive-free and dissolves rapidly in TBS or PBS buffer at room temperature.
Can I use sodium metavanadate instead of proto-vanadate?
A: The two are not the same thing. Sodium protofarnesate, also known as sodium orthopantothenate, inhibits phosphatase. This protects the phosphorylation site of the protein.
Can I retest phosphorylated proteins after stripping?
A: Yes, but care should be taken to keep the process at a low temperature. Phosphorylation is best left for the first time, unless specifically needed.
If a tissue is to be tested for phosphorylated protein kinase levels, must it be stored on dry ice in a -80°C freezer immediately after collection?
A: As much as possible, but you don't have to be so strict, it depends on the nature of your protein.
