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[KristinaZ]

General question about proteins tagged with small fusions: do you use a linker between your protein and a tag? What linker-how long and what sequence? Can the linker give me a problem? I added Flag to my retroviral vector (both N or C terminus) and I dont get expression of my fusion proteins. I sequenced the plasmid- all the sequences are correct and also my reading frame is correct. 

I put Flag into BamHI site and AICDA (my gene) into EcoRI and XhoI of pMX retroviral vector. I added some nucleotides to create a linker between  Flag and AICDA in hope that Flag would be separated by the linker and not obstructing my AICDA. However I have no protein expression  (Flag western). gca ggt gga  tcc gaa ttc  sequence predicts a cryptic splice site. I tried to make cDNA from HEK cells transfected with these constructs with oligo dT primer and PCR for AICDA but got nothing. 

Can you please give mew any advice?

 

 

 

 ggatcc gcc acc ATG GAC TAC AAA GAC GAT GAC GAC AAG gga tca gca ggt gga  tcc gaa ttc GAC AGC CTC TTG ATG AAC CGG

 

                          M   D   Y     K    D     D    D     D     K     G   S   A    G   A     S   E    F   D    S      L    L     M    N      R

 

 BamHI   Kozac                                             Flag                                     BamHI EcoRI               AICDA

 

                                                                                                                  -----------------------

 

                                                                                                LINKER

 

 

 

 

Sorry this is a cross post but there are many possible problems with the fusion proteins so I tried to pose another question.

 

 

Edited by KristinaZ, 10 October 2013 - 04:54 AM.

[dimensionsbio]

The length of the linker would depend on the downstream experiments that are to be done with the protein.  For example, if the protein is going to be immoblized for a binding assay, i would use a longer linker (8-10 amino acids) to attempt to create more space between the protein molecules to allow binding partners or ligands access to binding sites.  If the tag is simply for purification purposes or used as an epitope, then a linker may not be necessary.

 

I have had many isssues with anti-FLAG Western blots (not sure if that's what you are using to detect expression) and always run a control for the primary antibody on each blot.