I agree fully with Agata. Do as she has suggested.

I would like to know the native localization of this protein in order to be able to contribute anything further. Post-translational modifications (or their absence) are one big source of changes like these. If you let me know, maybe I can contribute something.

Those positively charged amino acids could be a major source of artefact. Histone and for that matter most highly basic proteins dont lend themselves well to SDS-PAGE. Also, remember that meracptoethanol (or DTT) cannot break all dimers. Sometimes even B-ME is partly oxidizsed.

Native localization of my protein is in cell surface.

More info about the protein: Mucus binding protein from L.acidophilus (gram +ve),

the protein has a 35 a.a. tail which includes LPXTG motif (carboxy sorting signal) followed by few hydrophobic a.a. and at last +ve charged a.a. which i mentioned earlier. Cleavage occurs between the Thr and Gly, that attaches the protein covalently to the cell wall. This kind of protein occur in almost all Gram-positive bacteria.

@Agata Thanks agata and i have already send the sample for MS and waiting for the results, but i am thinking that if possibly my protein has bind to some other protein then in MS analysis will it show the perfect match in MASCOT search?

Heavely glycosylated proteins show higher molecuar weight on SDS-PAGE; Ebola viiurs GP1 has an expected 75 KDa MWT and migrates on SDS PAGE as 150 KDa, Also some proteins with a high number of charged residues show abberrent migration on SDS page,. You can confirm the identiity of your protein using western bot if you have different antibody against your protein ( e.g anti his and anti your native protein). Alternative to that, you may do MS,

Somehow I could not understood the answer of Agata. DNA or RNA shift could be possible only with nuclear protein with its native structure. How denatured protein can interact with the DNA or RNA? Moreover, binding of protein occurs only with the specific consensus sequence of DNA oligonucleotides. RNA more suscetable to the RNAase which is present normally in non DEPC treated water. There could be some other specific reason(s). Glycosylation could be another possibility to increase the mol. wt. This can be assured by adding some chemical that can stop glycosylation during expression.

It could also be that you accidentailly purified GroEL, which is sometimes vastly overexpressed after IPTG addition. I once accidientally purified milligrams of it....

It might of course still be gylcosylation, though, I guess MS will reveal what is going on.

@Dennis : You mean to say that the purified protein would be GroEL or any other heat shock protein in response to my protein in the host? For control and out of curiosity i had given IPTG induction to cells having a simple vector (No gene insert) and further purified it and in that case their was not a single protein in elution visible after comasive stain. Though i have given for MS analysis but if my protein is interacting with other protein or tRNA then MS results would give wrong output?

@Paul : i think you are correct it might be HSP60. I have checked the insoluble fraction most of the protein is going in the insoluble fraction very less i am able to recover from soluble fraction. i am attaching the gel picture in which control is only vector without insert and mubp is my protein of interest. I have checked the flowthrough but no band of desired size.

And for MS i have cut the band precisely and send for the analysis.

This is 20C IPTG expression condition and thanks paul will try with 500mM NaCl. If this protein would be HSP60 than it should not be in soluble form?

I would also try high salt lysis buffer. If I remember correctly, it helped me out when I had the chaperone-problem. Also, you could try to add ATP during the purification, since it "opens" the chaperone - that might help as well and is easy to do. I prefered to do simple things first before screening all kinds of induction conditions etc.

@Agata I have tried glucose in the media at a time of inuction and in presence of glucose the protien was having little bit high expression but in the end same results i got. We dont have pLysE i need to arrange from somewhere and i will try in that also. I didnt try ethanol addition and but will plan to do that as per your suggesstion.

@Dennis: Would try ATP addition during lysis of cells. how much should i add ideally?

I found this document. The Hyman lab usually knows what it is doing... You use ATP during washing of your affinity column.

Thanks Dennis

Let us know if it worked!

Sure Dennis..

its good idea. but i don't think it will work in Ni affinity column.

then also you just do it

At first if u believe its your protein of interest try to purify it initially with ion exchange or other chromatography techniques then confirm it by MSMS.

This will help you to resolve your exact problem and then only u can make a good plan to purify it.

Perbhaps your protein is glycosylated

How do you know that your protein has a MW of 39KDa and not 60 KDa.There is no need to have doubt of its forming a dimer. But are sure that it is not. bound with some other ligands like carbohydrate or lipids. No I am not referring to glycoproteins or lipoproteins.

Most of the times the 60kDa band comes from up regulated GroES chaperon due to misfiled overexpreased protein. Also common is HSP70 (DNA K, 70kDa).

In rare cases a ATP wash resolves the problem, but from my experience better fragments or buffer conditions or expression systems are needed.

All the best.

Intrinsically disordered proteins very often possess abnormal electroforetic mobility. If you have a sequence of your protein you can analyze its disorder propensity by a number of computational tools. Here is a link to one of those: http://www.disprot.org/metapredictor.php

I agree with possible glycosylation. You can verify this with glycosidic hydrolysing enzyme follow by SDSPage again Since you found this mostly in the insoluble fractions then your protein could have folded uncorrectly Sometimes reducing the speeds of protein synthesis result in more soluble and correctly folded protein Westeen blot analysis will confirm your protein When you have more info on the aa sequence then you can have better picture of the 60 kd protein in comparison with the expected 39 kd Thank you all for the great discusion

Hi everybody...!!!!!!!!!!!!

I got the MS/MS results and its says that the band at 61kDA is mine protein of interest Mucus binding protein (39kDA). I am attaching the pdf result file.

Top score of MS is 71 for mucus binding protein and the next hit is 30S ribosomal protein S4 (22kDA) whose score is 68.

If i combine molecular weight of both since the result score 71 and 68 is not different i.e. 39+22kDA it comes to 61kDA. Is that my protein is interacting or making dimer with the other ribosomal protein and thus resulting into abarrent shift??

Did you boil the sample? In some cases this can cause oligomerization. A couple of things to try do not heat the sample after adding SDS-containing protein sample buffer, or add heated protein sample buffer to denature. You should also make sure that the you have sufficient reducing agent (TCEP works well). Many proteins do not need to be heated in the SDS buffer to fully denature.

You can try to put both proteins in the STRING database and see if there is a interaction possible.

It is not clear how these two proteins can be found together in your sample since they are from different organisms. Did you use Lactobacillus fermentum to produce

Mucus binding protein from Lactobacillus acidophilus NCFM? I would recommend to do sequence alignment for Mucus binding protein and 30S ribosomal protein S4.

I used Lactobacillus acidophilus NCFM to get Mucus binding protein but it is displaying fermentum result because the mascot hit has searched only firmicutes for possible hits so its showing the nearest matching that is Lactobacillus fermentum. I have checked alignment of 30S ribosomal protein S4 from fermentum and E.coli and it showed good matching so i guess that must be E.coli 30S ribosomal protein S4 where MUB interacts..Probably i dont know exactly

You should do some immunochemical analysis (western bloth) of your sample and use antibodies agains both Mucus binding protein and E.coli 30S ribosomal protein S4. This will be the most direct way to demonstrate that these two proteins interact.