Western Blot question

[theonlytycrane]

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Is there typically a minimum concentration of protein needed for detection? For the RAD51 control cases, for example, would it be valid to say that no RAD51 is present? Or could it just be in very low concentrations that the blot didn't pick it up.

 

[deleted585480]

I thought we only need to know southern blot? This wasn't covered in any of the Kaplan books?

 

[akimhaneul]

View attachment 207846

Is there typically a minimum concentration of protein needed for detection? For the RAD51 control cases, for example, would it be valid to say that no RAD51 is present? Or could it just be in very low concentrations that the blot didn't pick it up.

You are correct. There is no RAD51 for control. Even if there was some, it is highly insignificant compared to RAD51 overexpressed condition, so you can confidently conclude that control has no expression for RAD 51. This is why actin bands are important. Actin is a good loading control for western blots. It is present all the time so if you don't get a band for something while you get a nice actin band, it allows the scientist to show that western blot process is working well, they did not use different amount of protein for different conditions, and that the protein you are testing is not there.

 

[theonlytycrane]

@JPS398

 

[deleted585480]

@JPS398

View attachment 207850

thank you if you have any other neat memorization things hit me up haha!

 

[laczlacylaci]

You are correct. There is no RAD51 for control. Even if there was some, it is highly insignificant compared to RAD51 overexpressed condition, so you can confidently conclude that control has no expression for RAD 51. This is why actin bands are important. Actin is a good loading control for western blots. It is present all the time so if you don't get a band for something while you get a nice actin band, it allows the scientist to show that western blot process is working well, they did not use different amount of protein for different conditions, and that the protein you are testing is not there.

From your post, I understood that actin bands seem to act as an experimental control (if its working or not) for Western Blotting. Are there similar bands that can be applied to Southern and Northern?

 

[aldol16]

Yeah, so it's possible that your protein of interest simply isn't expressed enough to be detected. This is a good thing to keep in mind when you're actually running experiments. Actin or GAPDH is a good housekeeping tool but keep in mind that that only determines whether your sample is concentrated enough. If your sample is not concentrated enough, the Western wouldn't detect any protein even if those proteins were normally expressed in cells. But you're more concerned about whether your specific protein is expressed at sufficient quantities in the cell.

 

[andelJ94]

I thought we only need to know southern blot? This wasn't covered in any of the Kaplan books?

The AAMC only explicitly mentions 1 blot, that does not mean other techniques cannot come up or be asked about. I've already seen this in my AAMC materials.

 

[deleted585480]

The AAMC only explicitly mentions 1 blot, that does not mean other techniques cannot come up or be asked about. I've already seen this in my AAMC materials.

gotcha. seems fair.