What experiment could you run on fractions collected by diff

What experiment could you run on fractions collected by differential centrifugation of ribosome subunits to determine whether the polypeptide chains attached are on the small or large subunits?

Solution

Answer:

We can confirm and differentiate whether the polypeptide chains attached are of small or large subunits are:

1. SDS-PAGE:

Running the samples on SDS-PAGE can give the characteristic bands and patterns of proteins which constitutes either the small or large subunits.

If the isolation of the ribosomal subunits is done from translating ribosomes, other accessory factors/proteins can also be detected. The sequences of the individual bands can be confirmed through MS-MS analysis.

2. Isoelectric focusing and 2D gel electrophoresis:

The samples can be run on 2D gel and after separation of the bands, they can be sliced out and MADI-TOF can be performed to know the sequences. Identification of the protein sequences can reveal whether they belong to the small or large subunits.

3. Cryo-EM:

If time is not the limiting factor, Cryo-EM can be performed to delineate the architecture due to the combination of polypeptides. Generally a high resolution structure can be obtained with Cryo-EM. The overall topology of the large molecule along with the sequences of the proteins and rRNA involved can reveal whether it is the smaller or larger subunit.

If time is a limiting factor, a crude method can be negatively stained EM which can reveal the corresponding envelopes.