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Flag fusion protein not detected by Flag antibody

retroviral vector expression in HEK cells

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8 replies to this topic

#1 KristinaZ

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Posted 01 October 2013 - 05:00 AM

Pmnx.jpg

 

 

Hello,

 

I am basically reposting this topic in a different section. Have you ever encountered this- I am having a problem to detect a fusion protein with the antibody to the tag. My protein is Flag-AICDA and I see the protein with anti-AICDA antibody but not with the Flag. 

 

This is the sequence of the fusion junction:

 

 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

 

My coleagues suggested that the fusion protein is translated from AICDA 6th aminoacid which is methionine.....but there is no Kozac sequence there-could this happen?

 

I need to use the tag for detection becuase I am going to study splice variants of this protein and the commercial antibodies are raised against the deleted regions. 

 
Do you have any suggestions how to fix the problem? my vector is pMX retorviral vector - map attached. Flag is in BamHI and AICDA in EcoRI-XhoI sites. My transfections with this vecotr quite poor in HEK cells.
 
Can you suggest  a better vector - even YFP would be good.
 
Thank you!

 

 

 



#2 bob1

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Posted 01 October 2013 - 11:47 AM

I'm no expert on this, but it is possible that the extra methionine is acting as the start site and the sequence is being transcribed from there, but this is unlikely unless the promoter in the plasmid is less than 30 bp away from your kozak/atg site.

 

It is more likely that there is some conformational issue, such that the flag (despite it's high hydrophilicity index) is being tucked away inside the protein so that it isn't detectable from the outside.  This should only really be a problem for things like immunocytochemistry and should not apply to denaturing SDS-PAGE systems.  It could also be that there is some sort of splice site or digestion site in the protein that is causing the flag to be removed.

 

Are you sure that the flag antibody is working (i.e. have you tested a known flag +ve)?



#3 Missle

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Posted 02 October 2013 - 04:36 AM

For confirmation, you could try denaturing your sample with something stronger than SDS (urea?) and then probe with the anti-Flag antibody.  If you have binding then you know there is a steric hinderance thing going on but if you still don't then it is most likely that the Flag tag isn't there.  The premise of Flag is that it can be detected readily and is likely to be on the outside of your protein.

 

I agree with bob1 that a positive control Flag protein will provide clarity as well and that it is unlikely to get transcription at an internal methionine that is so far away from your promoter......

 

Have you used this particular anti-Flag antibody before?  Who makes it?



#4 KristinaZ

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Posted 02 October 2013 - 05:05 AM

Hello,

 

thank you for your response. Yes my Flag antibody works well - it even works on the same AICDA inserts cloned into expression pcDNA3.1 vector with C terminal Flag. The funny thing is that these inserts don't work with C terminal Flag in pMX. Is it possible that there are some splice sites generated in the pMX vector by Flag addition so that the Flag is spliced out?

 

Promoter and Kozac should be far away since there is psi retroviral sequence in between.

 

 

Thank you.



#5 KristinaZ

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Posted 02 October 2013 - 05:10 AM

Do you know a good commercial retroviral vector with a fluorescent tag that I could use to reclone this? 

 

Thanks



#6 KristinaZ

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Posted 02 October 2013 - 05:12 AM

When I use SDS the protein is not completely denatured? I thought it would be and I never had a problem with with FlagAID in another vector (thought cloned differently). I know that I created a few extra amino acids in the linker between Flag and AICDA but I thought that they would be denatured so it should not matter. Any idea?


Edited by KristinaZ, 02 October 2013 - 05:13 AM.


#7 Missle

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Posted 02 October 2013 - 06:59 AM

Sorry but I don't have any experience with fluorescent constructs.  SDS generally completely denatures a protein but not always....sometimes additional denaturation is necessary. 



#8 IBMSterry

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Posted 09 October 2013 - 10:46 PM

hello,

  i have came across this problem, too.  at last, i replaced the single tag with 3xflag, and it always works well.

  best wishes.



#9 KristinaZ

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Posted 10 October 2013 - 04:37 AM

Thank you for advice. The problem with 3xFlag is that it is 3x bigger than 1xFlag. I need a very small tag. Big N terminal tags impair AID protein import. I cannot use C terminal tag because 2 of the  AID splice variants have stop codons.Do you think that cloning HA tag instead of Flag would help? I could just cut out the Flag (in BamHI site) and ligate annealed oligos for HA tag. 

 

I am doubting my linker. 

 

 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

 

This junction  gca ggt gga  tcc   where my linker is connected to BamHI site gives me a cryptic splice site in 2 predictor webpages.... Have anybody ever encountered this? Can my cloned sequence be spliced out? I tried to test this  by making RNA from the transfected cell (maybe 15-20% cells were transfected). I made cDNA with oligo dT primer and then PCRed with gene specific primers. However there is no product. Is it just problem of sensitivity? I have the same AID splice variants cloned in 2 different vectors (expression-pcDNA and retroviral-pMX). WEsterns on pMX-transfected cells are blank for Flag and very very weak for AID. Westerns on pcdNA3.1 transfected cells are fine with both Flag and AID antibodies. I tried N terminal and C terminal Flag in pMX but never detected proteins by western with Flag (Ab works well). Does anybody have any idea how to fix this? Half of my postdoc grant is supposed to be done with these clones and I worked on this cloning for 6 months already.... 

Sincere thanks for any advice. 







Also tagged with one or more of these keywords: retroviral vector, expression in HEK cells

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