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Cloning & Expression Problem in HEK-293Ts

Cloning Expression Myc HEK-293 Frame

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#1 IXA

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Posted 24 September 2012 - 07:00 AM

Hi everyone,

I've been following this forum for a while (and it has always proved useful) but this is my first post on the site.

I've been having numerous problems cloning and expressing a gene of interest in a pCMV-Tag3a mammalian expression vector for quite some time now.

I've had my construct sequenced and the N-terminal myc tag is in frame with my protein of interest.

However, upon transient transfection into HEK-293Ts, I am unable to detect expression the construct through Western blotting. I use a pCMV-Tag3a mammalian expression vector containing another protein (generated by someone else in the lab a number of years ago) as a positive control and can detect this through Western blotting with no problems. So I do not feel I have an obvious transfection issue.

I'm not quite sure where to go from here...

Appreciate any advice or help.

Thank you.

#2 bob1

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Posted 24 September 2012 - 12:40 PM

Welcome.

A few things - first check that your plasmid is OK - run some on a gel and make sure that it is largely intact and not degraded.
Could the gene be a membrane associated protein? If so, you may need to change how you solubilize the cells when making lysates. Try lysing in 2x Laemmli buffer and boiling straight away.
How strong is the reducing buffer for your running?
Do you see any aggregation of proteins after boiling the lysate?
How big is the protein? (what gel% are you using?)
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#3 IXA

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Posted 24 September 2012 - 02:35 PM



Welcome.

A few things - first check that your plasmid is OK - run some on a gel and make sure that it is largely intact and not degraded.
Could the gene be a membrane associated protein? If so, you may need to change how you solubilize the cells when making lysates. Try lysing in 2x Laemmli buffer and boiling straight away.
How strong is the reducing buffer for your running?
Do you see any aggregation of proteins after boiling the lysate?
How big is the protein? (what gel% are you using?)



Thank you for your reply. I'll try to provide more detail.

1. Plasmid has been run on an agarose gel and appears intact.

2. It is a membrane associated protein - it is a GPCR. I'm currently using RIPA buffer for cell lysis - I've tried lysing directly in 2x Laemmli buffer but to no avail. I've been advised that GPCRs may require a lower boiling temperature - perhaps this is worth a go too?

3. I'm using 2x Laemmli buffer (Sigma).

4. I do not see any aggregation after boiling.

5. It's approximately 50 kDa - running on a 10% Bis-Tris in MES so should be able to see the protein.

Thanks again for your help - much appreciated.

Edited by IXA, 24 September 2012 - 02:38 PM.


#4 bob1

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Posted 25 September 2012 - 12:34 AM

Ok, that all looks fine - I would try the lower temp thing, some proteins are really sensitive to temperature. Try 70 C for 10 min to start. I just found a protocol that says 65 for 5 min too.

Have other people transfected GPCRs successfully before? Could you detect the protein directly (i.e. do you have a GPCR ab) rather than via the tag?

#5 IXA

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Posted 25 September 2012 - 02:54 AM

Thanks again for your reply.

1. I shall try a range of lower temperatures/times with the boiling temperatures. Am I right to assume that RIPA buffer is suitable for GPCRs?

2. I'm not aware of anyone in the lab who has transfected GPCRs before - but will try to find out from other labs within the building.

3. Unfortunately I don't have a GPCR antibody or a way of detecting the protein directly!

Thanks again.

#6 bob1

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Posted 25 September 2012 - 12:57 PM

Thanks again for your reply.

1. I shall try a range of lower temperatures/times with the boiling temperatures. Am I right to assume that RIPA buffer is suitable for GPCRs?

2. I'm not aware of anyone in the lab who has transfected GPCRs before - but will try to find out from other labs within the building.
Thanks again.

1) no idea, it is suitable for most western applications though.
2) check out papers for GPCR expression off plasmids...

#7 rmbio

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Posted 19 July 2013 - 02:34 AM

Hi IXA...

Could you get the expression of GPCR in HEK293 cells? I am having the same problem posted here.
Please advise..

Thanks :)





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