Need technique to know which protein isoform is expressed in Dro. muscle?

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S.A.D.
S.A.D.'s picture
Need technique to know which protein isoform is expressed in Dro. muscle?

I'm working with the Drosophila larvae as my model system.

My gene of interest makes five predicted polypeptide isoforms and I am trying to determine which of these isoforms is being expressed in the larval muscle cells. All these isoforms differ from each other in only the C-terminus, otherwise they have identical sequences. Their MW are in range of 115-130 kDa.

Does anyone have a good idea of what technique I can use to identify which of these five isoforms is being expressed in the muscle?

I tried Western Blotting, but the Ab we have recognises the N-termius, and it doesnt discriminate one isoform from another. I just see one single band. Someone else had mentioned PCR but I dont know how exactly to go about it.

Anyone have a good strategy and/or protocol?

Thanks

mchinmoyee
mchinmoyee's picture
Identifying & isolating the

Identifying & isolating the muscle cell and then isolating the protein (specific isoform if its only found abundant) and purifyinng it for further analysis.
Or
Try running a 2D gel and recognise with same anibody you may get different spot if they have different pI and can easily isolate them from gel for further analysis
Or
You can tag your protein or related gene with GFP or other variety.
Or
Isolate and purify all isoforms and custom your antibody.

Good Luck

S.A.D.
S.A.D.'s picture
thanks for your response.

thanks for your response.

Can you elaborate on some of the options that you suggested. Basically I dont have the protein cloned or dont have it in purified form either.

All I do is disset the fly larvae, get the muscle layers, and boil them in loading buffer, subject to 1D electrophoresis, and then have been doing Western Blots (with Ab that recognizes all isoforms). So given this scenario what do you think I could do. In specific could you elaborate on options 2 and 3 (2D gel and GFP tagging)

Thanks for all your help

mchinmoyee wrote:

Identifying & isolating the muscle cell and then isolating the protein (specific isoform if its only found abundant) and purifyinng it for further analysis.
Or
Try running a 2D gel and recognise with same anibody you may get different spot if they have different pI and can easily isolate them from gel for further analysis
Or
You can tag your protein or related gene with GFP or other variety.
Or
Isolate and purify all isoforms and custom your antibody.

Good Luck

mchinmoyee
mchinmoyee's picture
Refer this paper I just came

Refer this paper I just came across, it will guide you to large extent.

1. Isoforms of a cuticular protein from larvae of the meal beetle,
Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis
S. HAEBEL, C. JENSEN, S. O. ANDERSEN and P. ROEPSTORFF
Protein Sci. 1995 4: 394-404

2. Size and charge heterogeneity of C-protein isoforms in avian skeletal muscle. Expression of six different isoforms in chicken muscle.
H. TAKANO-OHMURO , S. M. GOLDFINE , T. KOJIMA , T. OBINATA , and D. A.FISCHMAN .
Journal of Muscle Research and Cell Motility 10, 369-378 (1989)

Guy Sovak
Guy Sovak's picture
If you have the possibility

If you have the possibility to run a 2D IEF try it as the results could be seen quickly.
That say the system should be calibrated and you should have a good antibody. It also depends upon how much protein you usually have in a sample.
Let us say in 75ug how much of your targeted protein will you have?
Guy