10 replies to this topic

[Biochemist25]

Hi, I have expressed two constructs. One just a GFP as a control and other mygene-GFP. After induction, under the microscope the cells with just GFP are green but mygene-GFP cells are not green.

Then I did a western blot using anti GFP antibody, but I don't see GFP band from GFP alone construct let alone the fusion construct:
Protocol:
(1) blocked membrane using 5% BSA PBST 1hour, RT with shake
(2) 3x 5min wash with PBST
(3) primary antibody (Abcam mouse GST antibody), 1400x dilution, incubated with shake in 4C overnight
(4) 3x 5min wash with PBST
(5) secondary antibody (santa cruz donkey anti-mouse IgG HRP), 5000x dilution, incubated with shake, RT, 1hour
(6) 3x 5min wash with PBST
(7) Mixed 0.5mL each of Pierce ECL Western Blotting Substrate and poured 1mL mix onto membrane
(8) Exposed membrane for 3 min

Cells are green before lysis and after lysis in western blot the lysate shows no GFP band. Either, western blot protocol is wrong or the GFP antibody is not working...

Please suggest any changes in the protocol if required... Thank you.

[laurequillo]

Biochemist25, on Feb 26 2010, 12:52 PM, said:

Hi, I have expressed two constructs. One just a GFP as a control and other mygene-GFP. After induction, under the microscope the cells with just GFP are green but mygene-GFP cells are not green.

Then I did a western blot using anti GFP antibody, but I don't see GFP band from GFP alone construct let alone the fusion construct:
Protocol:
(1) blocked membrane using 5% BSA PBST 1hour, RT with shake
(2) 3x 5min wash with PBST
(3) primary antibody (Abcam mouse GST antibody), 1400x dilution, incubated with shake in 4C overnight
(4) 3x 5min wash with PBST
(5) secondary antibody (santa cruz donkey anti-mouse IgG HRP), 5000x dilution, incubated with shake, RT, 1hour
(6) 3x 5min wash with PBST
(7) Mixed 0.5mL each of Pierce ECL Western Blotting Substrate and poured 1mL mix onto membrane
(8) Exposed membrane for 3 min

Cells are green before lysis and after lysis in western blot the lysate shows no GFP band. Either, western blot protocol is wrong or the GFP antibody is not working...

Please suggest any changes in the protocol if required... Thank you.

You know that GFP alone is quite small right? Maybe you let it go out. Anyway if you cannot see your protein in IF I would say that you are not expressing it

[Biochemist25]

Quote

You know that GFP alone is quite small right? Maybe you let it go out. Anyway if you cannot see your protein in IF I would say that you are not expressing it

Any reason why the GFP-tagged construct would not express. I have sequenced it and it's alright. The induction is under GAL promoter for both constructs, grown in parallel.

[laurequillo]

Biochemist25, on Feb 26 2010, 02:15 PM, said:

Quote

You know that GFP alone is quite small right? Maybe you let it go out. Anyway if you cannot see your protein in IF I would say that you are not expressing it

Any reason why the GFP-tagged construct would not express. I have sequenced it and it's alright. The induction is under GAL promoter for both constructs, grown in parallel.

No idea. Sometimes I have some clones that the sequence is ok but then I am not able to express them. The GFP tag is in N or in C terminus? sometimes the position of such a big tag affects the expression of the protein

Edited by laurequillo, 26 February 2010 - 06:24 AM.

[dpo]

Biochemist25, on Feb 26 2010, 12:52 PM, said:

(3) primary antibody (Abcam mouse GST antibody), 1400x dilution, incubated with shake in 4C overnight

I presume this is a typo? Otherwise it's quite clear why you see no signal...

[scolix]

Have a positive control if possible.

GFP can be detected easily on WB. Try new antibody aliquot of both primary and secondary. At times, the antibody has degraded and one never gets any signal.

good luck

[CambridgeBiochemist]

dpo, on Feb 26 2010, 04:43 PM, said:

Biochemist25, on Feb 26 2010, 12:52 PM, said:

(3) primary antibody (Abcam mouse GST antibody), 1400x dilution, incubated with shake in 4C overnight

I presume this is a typo? Otherwise it's quite clear why you see no signal...

Yes, that's GFP not GST




Note CambridgeBiochemist = Biochemist25 (for some technical reason I am not able to log in using Biochemist25

[CambridgeBiochemist]

laurequillo, on Feb 26 2010, 02:23 PM, said:

Biochemist25, on Feb 26 2010, 02:15 PM, said:

Quote

You know that GFP alone is quite small right? Maybe you let it go out. Anyway if you cannot see your protein in IF I would say that you are not expressing it

Any reason why the GFP-tagged construct would not express. I have sequenced it and it's alright. The induction is under GAL promoter for both constructs, grown in parallel.

No idea. Sometimes I have some clones that the sequence is ok but then I am not able to express them. The GFP tag is in N or in C terminus? sometimes the position of such a big tag affects the expression of the protein

The GFP is part of the pAG426GAL-EGFP-ccdb vector. It's N-terminal

[laurequillo]

CambridgeBiochemist, on Feb 26 2010, 07:36 PM, said:

laurequillo, on Feb 26 2010, 02:23 PM, said:

Biochemist25, on Feb 26 2010, 02:15 PM, said:

Quote

You know that GFP alone is quite small right? Maybe you let it go out. Anyway if you cannot see your protein in IF I would say that you are not expressing it

Any reason why the GFP-tagged construct would not express. I have sequenced it and it's alright. The induction is under GAL promoter for both constructs, grown in parallel.

No idea. Sometimes I have some clones that the sequence is ok but then I am not able to express them. The GFP tag is in N or in C terminus? sometimes the position of such a big tag affects the expression of the protein

The GFP is part of the pAG426GAL-EGFP-ccdb vector. It's N-terminal

And you said that you were not able to detect GFP alone right (GFP alone is about 27 kda)? try with another protein-gfp. You first need to know that everything is working in your WB

[ursae]

wad do u mean detect gfp alone?

Whr is ur protein localised? I had an experience in which my protein+gfp doesnt shown up in WB no matter what. Then it occurred that this protein is localised in the nucleus and my ripa buffer wasnt strong enough to lyse the nuclear membrane. After i switched to a strong lysis buffer, tada it showed up just fine.

Or other thing is that ur transfection didnt really work anyway.

Lastly, try to use another antibody from another company. ur ab might have spoilt/degraded. tat usually happens.

[laurequillo]

ursae, on 18 July 2010 - 06:04 PM, said:

wad do u mean detect gfp alone?

Whr is ur protein localised? I had an experience in which my protein+gfp doesnt shown up in WB no matter what. Then it occurred that this protein is localised in the nucleus and my ripa buffer wasnt strong enough to lyse the nuclear membrane. After i switched to a strong lysis buffer, tada it showed up just fine.

Or other thing is that ur transfection didnt really work anyway.

Lastly, try to use another antibody from another company. ur ab might have spoilt/degraded. tat usually happens.

well, Ripa buffer is quite strong, and I guess your protein was not in all the nucleus, but in some insolube bodies (pml bodies...). Anyway if you do a SDS lysis and you cannot detect your protein something is wrong with the WB. Maybe the antibody. I would add a positive control and then you will know