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I was given the task of determining the percentage of the S-protein of the SARS-CoV relative to the total of its proteins from the attached image. However, I have been given no explanation of the image, and with a physics background, I simply do not understand it. Can someone please explain to me how it would be possible to solve the task from the given image (i.e., explain the image).

enter image description here

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I'm not going to give you the final answer, because this is still a class assignment, after all, but I'll give you some tips. What you are looking at is a Western blot of whole-cell extracts from a cell line called Vero, either on its own (vertical ane 9, from numbering across the bottom) or infected with various viruses carrying specific genes, as indicated. Lanes 1-7 are from Vero cells infected with the BHPIV3 viral vector (a live-attenuated virus that can carry genes from another organism). Lane 1 is just the viral vector itself, with its own proteins. Lanes 2-7 are from cells infected with the viral vector carry certain genes from the SARS coronavirus. Lane 8 is from cells infected with SARS-CoV itself (so it carries all the previous genes). The numbers along the far right side are molecular weights, in kilodaltons.

A Western blot is performed in three parts - 1) separating the protein samples (whole-cell extracts) by molecular weight (size) in an SDS-PAGE gel by using an electric field, 2) transferring the separated proteins to a membrane, also using an electric field, and 3) using some sort of probe (usually a specific antibody) to determine what is present and in what amount. In this case, two antibodies were used. The lower, narrow image shows the samples probed with an antibody that recognizes the HPIV3 F protein, which is present in all the BHPIV3-infected lanes, in relatively equal amounts. This can be determined because the size/intensity of the different bands in each vertical lane is directly proportional to the amount of that protein present in the original sample.

The upper part of the image shows the extracts probed with an antibody that evidently recognizes the SARS S, M, and N proteins, but apparently not the E protein (as Lane 6, with the SARS-E virus, is blank). Using this information, you can now determine which bands correspond to which protein.

Keeping in mind that Lane 8 contains all the proteins from the SARS-CoV virus, while the previous lanes just contain certain ones as labeled along the top, you should now be able to use ImageJ to answer the question. I will give you one hint - there is much more information present than you need to answer the question. You actually only need 4 (well, maybe 5) of the lanes. I'll leave it to you to figure out which ones.

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  • $\begingroup$ Thank you for your very elaborate answer; it helped a lot. One question. You say that lane 8 contains all the proteins from the SARS-CoV virus, but that I need 4-5 of the lanes to answer the question. But if lane 8 contains all the proteins, could I not get an estimate of the relative amount of the spike protein from this lane alone? $\endgroup$ – Physics101 Apr 29 at 10:33
  • $\begingroup$ @Physics101 You need the other lanes to tell you which band the S protein is, and to tell you which (if any) bands are background artifacts. The E lane is also important, as it affects how you report your results. $\endgroup$ – MattDMo Apr 29 at 12:39
  • $\begingroup$ Okay, thank you very much. $\endgroup$ – Physics101 Apr 29 at 13:18

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