I recently fused two ORF protein with IgG Fc fragment in pCAGEN vector. After transfection in 293T cell, using two mAbs of the ORF or the antibody against human IgG, I can observe obvious expression. But when performing WB, by the two mAbs against the ORF, I can see a specific band, but the band size is 15Kd smaller than the total size. While using the HRP-antibody against human IgG, I can't see any specific band. I checked the antibodies, they works fine against other protein.
The proposed size of ORF should be 35kd, and the size of Fc should be around 23kd. After sequencing, there is no stop codon in the sequence, only three AA mutation in Fc fragment. So I suspected the overexpressed protein maybe misfolded or processed by 293T cell, so the downstream of Fc is broken. Besides, I add a secrete signaling peptide upstream of the ORF, but the protein was not secreted to supernatant when I expressed it in CHO cell.
If there is any analysis or suggestions, I will be very appreciated!
The reason why HRP conjugated 2nd Ab against human IgG can not recognize your ORF-Fc fusion proteins in WB is due to lost of epitope. Each antibody recognize specific epitope that can be categorized into linear epitope or conformational epitope. An antibody that recognize linear epitope can be used in a western blot, because protein is now denatured and blotted onto membrane thus the linear epitope is exposed for detection. This same antibody maybe used in IFA if the linear epitope is available for recognition when positioned in a turn or loop. For antibody that recognize conformational epitope, it will not recognize a protein been denatured and blotted onto membrane in a WB, because protein has lost its conformational epitope. HRP conjugated secondary Ab against human IgG is only good when human IgG is an intact antibody with native conformation, but you have it in WB.
As for why these two mAbs against your ORF only detect a smaller specific protein in a WB, the predicted protein size may not reflected its position in WB. I think as long as you have a negative control without transfection for comparison, the band you see should be your fusion protein even though it may be smaller than you expect. OR the fusion protein is not translated from the very beginning, check if you put kozak sequence around the methionine you intended to start with. If not, translation machinary may skip the first ATG and start with downstream ATG.
The reason why HRP conjugated 2nd Ab against human IgG can not recognize your ORF-Fc fusion proteins in WB is due to lost of epitope. Each antibody recognize specific epitope that can be categorized into linear epitope or conformational epitope. An antibody that recognize linear epitope can be used in a western blot, because protein is now denatured and blotted onto membrane thus the linear epitope is exposed for detection. This same antibody maybe used in IFA if the linear epitope is available for recognition when positioned in a turn or loop. For antibody that recognize conformational epitope, it will not recognize a protein been denatured and blotted onto membrane in a WB, because protein has lost its conformational epitope. HRP conjugated secondary Ab against human IgG is only good when human IgG is an intact antibody with native conformation, but you have it in WB.
As for why these two mAbs against your ORF only detect a smaller specific protein in a WB, the predicted protein size may not reflected its position in WB. I think as long as you have a negative control without transfection for comparison, the band you see should be your fusion protein even though it may be smaller than you expect. OR the fusion protein is not translated from the very beginning, check if you put kozak sequence around the methionine you intended to start with. If not, translation machinary may skip the first ATG and start with downstream ATG.
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