B/B Section bank #62 (centrifugation)

[theonlytycrane]

The supernatant will contain the cytoplasmic contents. But does the pellet contain the organelle precipitates?

---

Members don't see this ad.

 

[laczlacylaci]

Do you mind explaining how you got to your answer? 🙂

 

[wizzed101]

The pellet is the precipitate. Just think of the supernatant as an aqueous solution. W/e is in the supernatant must be soluble in water.

 

[theonlytycrane]

@laczlacylaci

The passage talks about investigating whether pRB protein is involved in mitochondrial apoptosis. Cytochrome c is in the inter-membrane space of the mitochondria, so if the mitochondria undergoes apoptosis, cytochrome c could be found in the cytoplasm of the cell.

Figure 1 (in the passage) shows that Bax activates apoptosis. We see this because without Bax (-/-), the apoptosis levels are not significantly different than the WT.

Now we look at the question where pRB protein levels are either 0 or 500 nM and we either use Bak & Bax or not. After this, cells are spun around in a centrifuge and membranes are popped. Imagine the cell contents spilling out with the organelles being the pellets and the cytoplasm being the SN (supernatant). If mitochondrial apoptosis occurred, cytochrome c would be found in the supernatant as well as the pellet. If not, it should be localized to the pellet.

The largest stain for cytochrome c in the supernatant should be in the combination where pRB protein levels are 500 nM and we use Bak & Bax. Only (a) and (b) show this, but (b) also shows some cytochrome c showing up with pRB at 0 nM or in the case without Bak & Bax (which is wrong).

 

[laczlacylaci]

That makes more sense. I think I would have to read the passage to understand the full story more. Thank you!

 

[bobeanie95]

The largest stain for cytochrome c in the supernatant should be in the combination where pRB protein levels are 500 nM and we use Bak & Bax. Only (a) and (b) show this, but (b) also shows some cytochrome c showing up with pRB at 0 nM or in the case without Bak & Bax (which is wrong).

@theonlytycrane

For B, how did you get that the combination pRB(0)/BAX and BAk (+) and pRB(500)/BAX and BAK (-) should be completely zero, showing no levels of apoptosis? There's no data in the passages or figures to indicate that both, pRB & BAK and BAX, are absolutely required to visualize some level of apoptosis. In other words, the figures indicate that pRB is always held at basal or induced levels, so you cant assume levels of apoptosis will be zero when pRB is absent. Could you elaborate?

 

[theonlytycrane]

@bobeanie95 Sorry- I didn't mean to imply that the staining should be 0 for those cases, but the staining should be relatively less than in the pRB(0) case than pRB(500) and less without Bax + Bak than with Bax + Bak. So I looked for an answer choice that showed this trend, focusing on looking at the supernatant.

 

[bobeanie95]

@theonlytycrane Yes, I agree with you, which is why I couldn't come down between A and B since both showed decreased levels in lanes 1 and 4. Since the passage didn't provide the exact mechanism or data regarding lanes 1 and 4 (pRB(0)/BAX and BAk (+) and pRB(500)/BAX and BAK (-)). The passage never implies whether apoptosis levels are cumulatively affected by pRB and BAX/BAK or its dependent on both for apoptosis to undergo. I'm not sure how you could come to the conclusion to pick A over B.

 

[theonlytycrane]

@bobeanie95

Figure 1 indicates that apoptosis is Bax dependent, which makes me lean towards the answer showing apoptosis only when Bax + Bak is present.

 

[bobeanie95]

@theonlytycrane Right I see what you mean. I guess I reasoned it by the fact that even though its Bax dependent, there is still around 5-10% apoptosis in Bax-/- so there should still be some staining in the gel. I guess option A has this slight staining in lane 4?

Even though apoptosis being Bax dependent, this doesn't necessarily mean that apoptosis is pRB dependent. So it can't be said for sure whether lane 1 will be clear when pRB is absent, yet Bax is present.

 

[theonlytycrane]

The experiment investigates pRB protein (transcriptional coregulation) on apoptosis, via a Bax and Bak protein activation pathway.

The results aren't presented as though pRB and Bax/Bak are independent. Rather, that pRB induces expression of some proteins to induce apoptosis.

 

[bobeanie95]

@theonlytycrane great thanks!

 

[betterfuture]

Can someone give me a rundown on what organelles go where during centrifugation? What would be in the supernatant and pellet?

 

[laczlacylaci]

Can someone give me a rundown on what organelles go where during centrifugation? What would be in the supernatant and pellet?

In the supernatant would be the cytoplasm/cytsol and in the pellet would be all the organelles.
You can think of a cell like the organelles are the solutes/heavier stuff and the cytoplasm is the solvent.

From the passage, we know that BAX and BAK are activated in the presence of pRB (which is related to intrinsic/mitochondrial apoptosis), we will definitely expect to see some cytochrome c here (also according to the passage) The cytochrome c exists in the pellet (organelle: mitochondria) that is why we see the big blob. The reason why we see a blob under +pRB +BAX/BAK in the SN, is because cytochrome c can be released out of the mitochondria for apoptotic reasons.

A faster way to approach questions like this is the see the similarity of all 4 choices, you will notice that in the pellet section, all 4 are the same, so you just need to focus on the SN part.