I am going to do proteomics analysis from cancer tissues.
I am doing this for the first time. I am going to deal with solid tumour samples some time I will get fresh tissue. So, to do my job first I need to fix those sample and then decrosslinking.
Multiple ways for fixation is available like FFPE, UMFix for tissue and they also mentioned the way of decrosslinking but they having different advantages with some dis advantages.
I am looking for a new and effective method of tissue fixation which should not lead to loss of proteins just because of fixation or ineffective way of decrosslinking.
So, If anyone doing same kind of work or having experience with same kind of work please let me know the way how you are doing and how much effective it is.
Thanks in advance
Hi, Amit -
As Jason Lies suggests, can you give us some additional information? When you say you 'are doing this for the first time", what do you mean? Are you doing proteomics for the first time, soft-tissue experiments for the first time, or both of these for the first time. If the latter (i.e., both for the first time) what you're proposing is not trivial, and involves mass spectrometry, bioinformatics, and - as you indicate - tissue handling; and success will depend on skill and experience in all of these aspects.
One suggestion would be to work first with well-behaved standards (e.g., commercial albumin digests) before you attempt working with more-difficult (and more valuable) actual samples.
If you're experienced with proteomics, and just beginning soft-tissue experiments for the first time, I apologize for preaching to the choir or bringing the coals to Newcastle.
:)
Have a look into using antigen retrieval methods and use of 2,4-DNPH in mass spec, i found a very interesting paper a while back which I cannot find at the moment which has a great method.
I have done some work on using formalin for cross linking. Its also interesting to see how monomers react as well, I have a comparative spectral image on my page of how bovine beta-oxidized insulin reacts with formalin (4xamines present) which may help guide you. Unfortunately I cannot put much more data up due to IP.
As John Wishnok rightly points out this is not an easy task.
If you are working direct from sections in a shotgun proteomic manner, normal slides will not work. indium-tin oxide slides are needed for MALDI-TOF analysis due to the need for the stage to pass a current.
Hi, Amit
I have done my experiments on formalin-fixed, paraffin-embedded by using IHC, 1 D, 2 D immunoblot, and LC MS/MS. I got nice results but you have to find the correct protocol and the antigen retrievals important to avoid cross linkage as Jason mention.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics