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Protein repeatedly does not express from pMX retroviral vector-advice needed des

retroviruswestern blot HEK293 fusion-protein expressio transient transfection

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10 replies to this topic

#1 KristinaZ

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Posted 24 September 2013 - 07:03 AM

Hello. I hope to get an explanation why I should trash my 6-month long effort. I cloned a mRNA from a patient into pMX retroviral vector (as either C or N-terminal Flag fusion protein). When I transiently transfect HEK293 with this construct I do not detect my protein with the Flag antibody. The cells are transfected since the retorviral vector has a GFP (translated from IRES) and the cells are green (at least 30% of them). I also cloned this mRNA into pcDNA3.1+ vector and the expression is beautiful. What am I doing wrong? Can the protein form retroviral vecotrs be deteted from transient transfections?

 

I am eager to learn so please teach me. Thanks

 

KZ


Edited by KristinaZ, 24 September 2013 - 07:04 AM.


#2 jerryshelly1

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Posted 24 September 2013 - 01:33 PM

I never used this vector, but a quick google search indicates that the GFP and FLAG are at different promoters. Is your gene of interest in frame to the FLAG epitope tag?



#3 KristinaZ

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Posted 24 September 2013 - 10:25 PM

Thanks for the response. My pMX vector did not have a  Flag tag. I cloned it into the N terminus of pMX in frame with my gene of interest. I am working with splice variants of AICDA gene so I cannot use antibody to AICDA (would not detect the splice variants). 

 

 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    I      R

                            Flag                                                                            BamHI EcoRI               AICDA

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

 BamHI   Kozac                                                                                                 LINKER

 

This is my sequence that I inserted in pMX (Flag with linker into BamHI site) and then AICDA into EcoRI and XhoI site. In my eye it is in frame and there is Kozac for translation initiation.....

 

Do you see anything out of place?

 

Thanks!!!


Edited by KristinaZ, 24 September 2013 - 10:26 PM.


#4 KristinaZ

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Posted 25 September 2013 - 12:35 AM

   How can you attach files here? I see no attachement pin here...I woul dlike to attache the map pf the vector (we sequenced it). Thanks



#5 jerryshelly1

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Posted 25 September 2013 - 10:48 AM

Everything looks good. You can paste media by going to my media and uploading the attachment.  If it is a word file, use the clip board with the word symbol on it.

 

Is there any increase in mRNA levels? I wonder if this is prone to degradation, especially since you can see the GFP.



#6 jerryshelly1

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Posted 25 September 2013 - 10:50 AM

Maybe run your DNA sequence with your FLAG tag though a secondary structure predictor to see if a weird hairpin develops that prevents proper translation.



#7 KristinaZ

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Posted 26 September 2013 - 05:09 AM

I am sorry to be do dull but when I click on my media it only gives me the option to search MY MEDIA LIBRARY. How can I upload files from the desktop? Thank you. 

 

Also the GFP should be transcribed from LTR as a bicistronic RNA. LTR - AICDA (=my gene) - IRES - GFP.  RNA of my gene should be there becasue I can see GFP (but I have not tested that). 

Thank you for suggesting checking the seconday structure. The strange thing is that I cloned AICDA to pMX with C terminal Flag tag first and only after I did not get any expression by western blot I put Flag into N terminus of pMX and then cloned AICDA as N terminal fusion. Neither works. I got expression of AICDA as  C temianl Flag fusion from pcDNA3.1. If it was secondary structure issue it would be strange to get it with both N and C termianl Flag ad clearly the secondary structure will be different....

I am breaking my head but I am clueless. Thanks for suggestions.



#8 Tabaluga

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Posted 28 September 2013 - 12:18 PM

Using the classic skin of BioForum, I do get an attachment button when I reply to this post (not with the quick reply, but with the "reply to this topic" button. Whic device are you using for looking at BioForum ?

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#9 KristinaZ

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Posted 29 September 2013 - 02:15 AM

Hello!

 

I found that when I express my fusion protein FLAG-AICDA I can detect it with AICDA antibody but not with the Flag antibody. I put 8 amino-acid linker  between the tag and my protein...it looks like this:

 

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    I      R

                            Flag                                                                            BamHI EcoRI               AICDA

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

 BamHI   Kozac                                                                                                 LINKER

 

Why would you say the Flag tag is inaccessible to its antibody? I need to use the tag because I want to study AICDA splice variants and they cannot be detected with AICDA antibody.

 

Thank you!

 

P.S>  I dont; know which skin I am using for bioforum-how can I find out? I go to My media but the options are strange- it has attachment but only from Your media library and I don;t know what that is. There is only one pdf file there called GEL...



#10 KristinaZ

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Posted 29 September 2013 - 02:25 AM

Finally I figured how to attached the vector I cloned into.Pmnx.jpg

 

Could any good soul here figure out if the RNA of my gene of interest is bicistronic with GFP? I put Flag into BamHI site and my gene aICDA into ECORI nad XHOI. I will be glad for advice. Thank you!



#11 Tabaluga

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Posted 29 September 2013 - 02:44 AM

P.S> [/b] I dont; know which skin I am using for bioforum-how can I find out? I go to My media but the options are strange- it has attachment but only from Your media library and I don;t know what that is. There is only one pdf file there called GEL...[/font][/color]


At the very bottom of the page, go to Change Theme and change to Classic.

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Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 






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