how to interpret this WB result - (Jul/30/2018 )

Hi everyone,

 

I'm a newbie and I really need help to comprehend and interpret this WB result.

 

Referring to the attached result, I'm using TRPV4 agonist (GSK101) 100nM concentration.

Then, I used 2 different TRPV4 antagonists, which are HC067 and GSK219 in different increasing dosages.

The different dosages of antagonists did not affect LCN2 secretion from primary rat adipocytes culture.

 

Could it means that the lowest concentration used (HC067 500nM and GSK219 10nM)) is adequate to occupied the receptor since there is no difference in term of band intensities when compared to the higher concentration of antagonists (HC067 1uM and GSK219 100nM)?

 

Is there any other possible interpretation or conclusion that can be deduce from the result?

 

I welcome any feedback or comment from everyone.

 

Thanks a lot!!

 

https://drive.google.com/open?id=1KjgBKqUQTR3xOqcDfZOSpyg6fp3HEdkg

Any chance you can post a picture directly on here?

I'm sorry..I'm not sure how to post the picture directly here..

Can anybody suggest me how to do that?

When you scroll down the the reply box, there should be an image button on the same row as the bold, italics and underline controls. It looks like a square with a green dot part in the middle. On my controls it is below the text color and emoji buttons.

To attach image to a post, first select More reply options on the bottom of the page, and there you can attach files through the form.

After it is uploded, you need to click Add to post as well, to insert it into the answer field. Then the image would be attached.

Anyway your docs link leads to a document, that is not shared at the moment. You can either attach it or fix the sharing on Google Docs and try to link again.

 

Thank you very much for your guide and instruction.

I've attached the image here.

Hope to hear any feedback and comment about my WB result.

 

Thanks!!

Your interpretation would be one possible interpretation - another would be that your results are that the antagonists are not working. Based on the image showing that the  GSK101 shows a faint band, I would assume that this is how the other antagonists should also present. I see that the IC50 values for your drugs are much lower than what you are using, perhaps you need to be titrating in the IC50 range to see a more definite effect.

 

To make assumptions of working/not working you need to run a couple of controls - the most important is a loading control - you need to show B-Actin or something similar to show that you have loaded the same amount of protein in each lane. You have a positive and negative control.

Thank you very much Thelymitra pulchella for the input.

 

Sorry for my incomprehensible notes I wrote together with the image that causing misunderstanding.

GSK101 is the TRPV4 agonist...HC067 and GSK219 are the TRPV4 antagonists.

The bands that showing similar bands are the samples treated with both antagonists with various concentrations.

 

You are right about the negative and positive control..I shall look into that.

 

You are also right about the IC50 of the drugs. The starting dosage that I used (500nM for HC067 and 10nM for GSK219) is the dose that was documented in the articles that I cited.

I was told the reason why it was that high because they are using 5 times IC50 concentration.

Is that what is usually done?

 

If I'm using high dosage of antagonist in this experiment, what possibly could happen?

Will the high dose antagonist possibly affect the outcome as compared to the low dose since I'm not combining the treatment with agonist?