Do you check your Protein size only by SDS-PAGE? It is common that Proteins are migrating not exactly according to their mass on SDS so you can see your band at 75KDa even if you have correct size. If you can use MS analysis or size exclusion chromatography to see how your protein behaves. Do you cleave your Histag or not?

Mr. Jawed while doing the restriction digestion, what enzymes you have used. Histidine tags are very specific. I suggest you to try to run the PAGE gel with different molecular weight markers. Run the gel in duplicates, one using normal marker and the other using pre-stained marker. Use the second replicate and do western blotting for confirmation of your desired band.

Do you check your Protein size only by SDS-PAGE? It is common that Proteins are migrating not exactly according to their mass on SDS so you can see your band at 75KDa even if you have correct size. If you can use MS analysis or size exclusion chromatography to see how your protein behaves. Do you cleave your Histag or not?

Thanks to all.i did not use the conventional DNA cloning method,i was inserted my gene by using Homologous recombination method in between the two His tag by adding start and stop codon to my gene.I did not check my protein size by SDS-PAGE because i am unable to get the correct size. There is no cleavage size around the insertion site of my gene.you can find the map of expression vector in the attached file.

Hi, so how do you know you have 75kDa band? How do you check it? Do you say about size of you gene or size of your expressed protein? Why don't you send your plasmid to seqencing to check if your cloning was correct. Can you do that?

Try this:

Uninduced cells with vector

Induced Cells with vector

Uninduced cells without vector

Induced Cells without vector

also make cell free extract of each by lysis/centrifugation and run those eight samples on a gel.

Which samples show the 75kDa band?

You can also do a quick His-pull down from the lysates and see if anything stuck to the beads.

EDIT: use empty pET vector instead of no vector control if you want to keep the antibiotic in the control.

Thanks for your suggestions.Respected Mikolajka ,Chopra & Gajewski.I have checked my protein size by SDS-PAGE and i always get the band at 75 KDa.I have checked my vector and gene sequence several times.its in correct translational frame started from ATG of NcoI after RBS site,and if all of the amino acid will translated present in expression vector till stop codon it could not be the size of 75 kDa,its max size should be 57 KDa,because the exact size of my protein is 53 KDa and remaining 4 KDa is the weight of tags.you can find SDS pic of my Protein in the attached file.

Dear Jawed,

As Aleksandra said, in SDS-PAGE the proteins are not migrate only based on their mass wight, because of different behavior of hydrophobic and hydrophilic properties of amino acids in electrophoresis conditions. ie if you have to different proteins with exact number of the amino acids, but they differ in sequence, we do not expect that they sit on the same position on gel because of different characteristic of aa. so, if you are sure about the sequence of insert in vector and reading frame, keep on your experiments. I dont know what is your plan in downstream, but if you are still worried about the size, based on availability of specific antibody, you can check expressed protein by western blotting or if it has a specific substrate check with it.

okay respected guys thanks for your suggestion,if i will get some thing i will let you know.

Thanks

Rohail,

looks like genuine expression to me. However, some cells express indigenous proteins when induction condition are harsh so I recommend induction with cells only (empty pET can't encode for a 75kDa protein) if you keep having have problems later.

Try pull that "75kDa" protein down with NITA beads and estimate size in a secondary method, e.g. DLS or SEC.

best of luck!

S.

i guess, his tag also ads up to the size of your protein by 16-17 kda. Due to this you may get a shift in size of your protein. could you please confirm this?

Dear Respected guys,

After pull down,I have got same size of my recombinant protein after western blotting by reacting my peptide to the specific antibody.this size is may be due to the some post translational modification of protein.

Thanks for your suggestion and helpful answers.

The protein you are expressing might be interacting with another protein in the bacterial cell. Or if could be forming a dimer. Do you boil your samples before loading into the SDS Gels? Sometimes that can cause a mobility shift and depending on the protein boiling can increase or decrease the mobility. I would try a secondary method to to further characterize what you are seeing with something like size exclusion chromatography or dynamic light scattering. Good luck.

Yes i was boiled my samples for 10 min at 95 degree centigrade.after protein purification and staining i got the same size of band and also after western blotting,may be my protein did not have interaction with another bacterial protein.

Thanks n have a good time

I had some similar experience with a protein from leishmania. After challenging for a while we concluded that his difference between predicted protein size and its size in SDS-PAGE is related to characteristics of aminoacids content (hydrophobic/hydrophilic...). PTM is also responsible in many cases.