Hey All,
I have a peptide composed of various domains. I have the sequence of whole peptide and individual domain. i wanna express that whole peptide on T cell. After expression, i wanna run the western blot to check it successful expression on t cells. anyone know how should i determine the size of whole peptide and individual domain ???
Thanks
well im not sure if this answers your question
however, the formula used to calculate the coding capacity of DNA is as follows:
amino acids encoded = [(size of DNA (in bases))/3]
> size of DNA = 3 x number of amino acids encoded
> predicted size of protein = number of amino acids x 0.11kDa
> 0.11 kDa is the average molecular weight of an amino acid
Dear Waqas Nawaz,
Try this link:
https://www.sciencegateway.org/tools/proteinmw.htm
Best regards,
Thomas
Thomas M.C. Binder I tried this and others similar calculator before. The predicted size is about 67KDa according to the calculator . However, my band on westron blot appear in range of 100KDa. i repeat several time but i didnt get blot according to the prescribed size.
Hi Waqas, as you wrote, the predicted MWt is 67 KDa, but what you have detected on the westeron blot was 33 KDa more. Please dont forget firstly, it is an expected value, secondly, most proteins are glycosylated and if that the case of your protein, then, you would expect to see a 100 KDa protein on the westernblot. Please check the Mwt of your protein in other references (Papers, books) and let me know what they say. I have a recombinantly expressed glycoporotein (GPIIIa) which is normally expressed on the paletelets and on the endothelial cells as well. However, they are differently glycosylated, so, on the western blot paper, I had different Mwts. best regards,
As Younis said, don't forget the post translational protein modifications!
Best regards,
Thomas
Younis Skaik Dear i checked other reference papers, their sequence is same as mine one. The only difference is the vector, carrying the plasmid. Their blot came in the predicted size as calculated i.e 67KDa. However, i repeat several times adopting their protocol, furthermore, i also optimized various parameters. But still didn't got any publishing result.
Best regards,
Dear Wagas,
Some times you could use the same or a similar vector (plasmid) in different expression environments.
If you use this vector in an eukaryotic environment, you will find a lot of posttranslational modifictions, like complex glycosyslation.
If use this vector in a prokaryotic environment, you will find other modification but no complex glycosyslation.
Insect expression systems will produce other posttranslational modifictions.
Best regards,
Thomas
Dear Wagas,
First, the vector itself does not play a role in the molecular weight of your gene of interest, a vector is only vector. But please and kindly check again if you have some tags in your vector, which also should be expressed with the peptide, Tags like MBP, V5 Histidine, if yes, then certailny would the molecular weight be increased. One more question, could you please load the paper you read and found out that they have the same M.Wt as yours. thank you in advance, regards,
Younis Skaik Dear i checked, there is no tag in my vector. Sure, i upload the paper here. This is the reference paper which i followed. I am going to detect the CD3 zeta protein. Here in this paper they used lenti vector and i am using the AAV vector. I followed the same procedure as they do.
Thomas M.C. Binder Dear we have the same expression environment. They also used HEK293t cell to express their gene. which is derived from human embryonic kidney cells.
best regards,
Dear Waqas,
Are you sure, that you use this cell line (HEK293t) really?
In a lot of laboratories, we have cross contaminations with other cell lines, like HeLa cells.
Best regards,
Thomas
Dear Waqas, I did look at the paper. The construct they used is similar to yours but not identical, the tail of your and their construct is identical (CD28-4-1-BB-CD3 Zeta), but i think the head of the construct is different. Could you please check it again. Thanking you, regards,
Younis Skaik Dear i check it thoroughly. The extracellular domain (head) is also the same. They used Hu5a8 antibody and mine is also same. one more thing, according to the calculator (https://www.sciencegateway.org/tools/proteinmw.htm) the predicted MW is about 63KDa. But they mentioned in the paper , that from the sequence it should be around 67KDa. why there is a little bit difference of 4KDa come from ? and how they calculate the predicted size ?
Thanks ,
regards.
did you use the same antibody in the western blot as they did use? thank you and my best regards
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