Hi Ewirghol

Just a question to how you process your samples: do you add DTT or beta-Mercaptoethanol to your lysis buffer SDS page running buffer? I ask this because TGFb dimers are linked by disulfide bonds. Adding DTT or bMercapto and boiling your samples will reduce the bonds and the main form of TGFb will be the 12.5kD form!

I hope this will help

Tobi

Hi Mr Tobi,

Thank you so much for your answer. your answer gave me so much hope.

I was wandering if the urea or the sonicating process have some effect on this.

I used the remaining protein samples after RNA extraction form injured mouse skin according to the protocol from this paper: .  

samples were washed with 0.3M guanidine hydrochloride in 95% ethanol, then  1:1 solution of 8M urea(in Tris-HCL,pH 8) and 1% SDS  was added and then sonicated strongly.

Electrode Buffer for SDS-PAGE : 3 g Tris, 14 g Glycine, 1 g SDS in 1L H2O .

So do you think the disulfide bonds were reduced by guanidine hydrochloride , urea and sonicating process ? this is why only 12.5 kDa was observed right? 

Do you know that  any paper is related to this? 

If possible, I  also would like to hear your advise about the the 44kDa(around 50kDa)  band.  We isolated the total protein from human skin cells(no DTT in lysis buffer), and used the same electrode buffer. TGF-beta1 was expressed at 44kDa and 25kDa(44kDa is almost 2 times lighter than 25kDa). I was thinking that 44 is unactivated from, 25 is activated. but after reading many papers, now I am confusing about witch band should I trust.

Hope to hear from you again. 

Thank you so much.

I don't know much about TGF but I have come across this kind of thing before with other proteins. It is possible you may be getting covalent cross-linking perhaps due to side-chain oxidation (which might happen in skin)? This can convert side-chains to aldehyde or ketone groups (carbonylation) which can attack lysine side-chains to form adducts. You could try to stain with aqueous coomassie and excise the bands for tryptic digestion and tandem MS. The peptide pattern would be identical if its a dimer but differ if its a precursor-post type relationship.

I hope this helps

Dave

If I dont remember wrong it could be around 12.5- 13 kDa.  But you can see it around 25 -45 kD weight on gel if you can not select the marker to see small differences of isolated protein. Good luck :)

 TGFbeta1 is, strictly speaking 12.5KDa, and on a denaturing PAGE/Western Blot you should see it at that weight. It usually exists as a dimer -25 KDa. The 44KDa form is probably TGFBeta/ latency associated peptide (LAP). This is actually the gene product and initially it is how it exists in the cell, but before secretion it is cleaved and re-associates. In this form it is considered inactive; in fact it does have some activity but nothing like "active" TGFbeta.

Its  protein with Multi functional in human body, for example controls proliferation, differentiation and other functions in many cell types. It was synthesize in many cells and have specific receptors in that cells. It positively and negatively regulates many other growth factors. It plays an important role in bone remodeling as it is a potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation and differentiation in committed osteoblasts. in tumar cell its molecular weight around (95, 50 and 25 KDa, manly the first one is non active.

Dear

I think you denaturation the TGFb by added SDS or B-mercaptoethanol and that gave you two subunit each one with mol.weight 12.5 KD. the molecular is around 25KD.

Thank you and goodluck.

How many kDa is TGF-beta?. Available from: https://www.researchgate.net/post/How_many_kDa_is_TGF-beta [accessed Jul 13, 2017].

The 12.5 would likely correspond to the monomer particiularly if you were using reducing conditions that would dissociate the mature dimer.  The protein is translated from mRNA to produce a latent form that is larger (-412 aa) so that may be what you are also observing.

Hugh  

I have the same question. My guess is that TGFb, LAP and LTBP are kept together by S-S bonds, so using reducing conditions would lead to 12.5KDa bands, i.e TGFb mature monomer, regardless if you have cells or ECM. Unless the antibody reacts with the LAP. Bands in the range 37-45 suggest the detection of LAP. However if the antibody is specific for the active TGFb, and does not react with LAP, we can assume that a band of 45-55 KDa is actually the TGFb+LAP, which in fact are bond covalently before the intracellular processing in the Golgi.