Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!

cell 293's Content

There have been 11 items by cell 293 (Search limited from 16-July 18)


Sort by                Order  

#173241 Different drug treating time

Posted by cell 293 on 05 February 2015 - 07:46 PM in MTT, Proliferation and Cytotoxicity Assay

I don't know how to choice the time point for testing cytotoxicity of drug.

 

Do i just choice the time point that i can see dose-dependent?

 

Can different time point represent the acute or chronic toxicity?




#173215 Different drug treating time

Posted by cell 293 on 04 February 2015 - 11:45 PM in MTT, Proliferation and Cytotoxicity Assay

Hi, everyone

 

To determine cell viability with MTT assay, many time points (6, 12, 24, 48 hours) with drug treatment are detected.

 

What is meaning of different time with drug treatment? 

 

 

Thanks a lot




#171337 cell viability with PI staining

Posted by cell 293 on 20 October 2014 - 06:55 PM in Flow Cytometry

Hi, 

 

I want to test the cell viability by PI.

 

But i have only found the protocol of cell cycle.

 

In the protocol, it have to add 70% EtOH to fix cell and add RNase A with PI staining solution.

 

Should i do the steps if i just want to test viability?

 

Should i do a trypam blue stain and visually counting to compare it?

 

Thanks a lot.




#171151 How to treat HEK293 with S9 fraction?

Posted by cell 293 on 07 October 2014 - 06:59 PM in Tissue and Cell Culture

Hi, everyone

 

In my experiment, i need to treat HEK293 cells with drug including S9 fraction.

 

In the protocol of S9. The medium including S9 have to remove and wash with PBS after treating in 4 hours.

 

But the adhesion ability of HEK293 cells are very week.

 

The cells will be lost if i wash them.

 

What can i do to improve it?

 

Thanks a lot.




#170748 Problems of beta-actin

Posted by cell 293 on 11 September 2014 - 08:13 PM in SDS-PAGE and Western Blotting

Hi, everyone

 

This is beta-actin. I got multi-signals about this. I am sure i didn't move the film when it was exposed to signal.

 

01.JPG

 

Was too much sample loaded? 

 

The same samples were used many times. It is no problem when i use it first time.

 

Was the store method wrong? I store sample in -20°C.

 

 

Thanks a lot.

 




#170583 Problems of western signal

Posted by cell 293 on 03 September 2014 - 07:20 AM in SDS-PAGE and Western Blotting

Thanks your reply

 

Do you mean i can prepare a membrane that is only with secondary primary? 




#170576 Problems of western signal

Posted by cell 293 on 02 September 2014 - 07:32 AM in SDS-PAGE and Western Blotting

Thanks for your reply.

 

I have  ponceau stained the membrane. It is no problem to transfer.

 

Do you mean to dilute primary antibody with non-fat milk in TBS?

 

Is it work to adjust the secondary antibody concentration?




#170571 Problems of western signal

Posted by cell 293 on 01 September 2014 - 09:11 PM in SDS-PAGE and Western Blotting

Hi everyone

 

I have problem to improve my weak signal.

 

I expect to gel the signal at 35, 50, and 103 kDa. In my data, i have to expose and get the signal around 10~15 min. But the signal is still unclear.

 

What can i do to improve it.

 

In this data, background is every high, but the signals what i want are weak.

 

1.jpg

 

 

In this data. How to get stronger signal? And how to removal the black point in the film?

 

2.jpg  

 

 

This is beta-actin. Sometimes i got two signals when numerous sample was loaded. I am sure i didn't move the film when it was exposed.

 

Is it normal? How to imporve it?

 

3.jpg

 

 

This is simple protocol of my western.

 

1. Dissolve cells by adding PBS and 2X sample buffer  and boiled directly.   (I don't lysis because protein will be lose.)

 

2. Start the electrophoresis process for the sample at 60 V.

 

3. Thansfer proteins at 300 mA, 2 hr.

 

4. Block membrane with 5% non-fat milk in TBS

 

5. Incubation with primary antibody (goat, 1:500) at 4℃ overnight (around 16 hr)

 

6. Wash 10 min with TBST (tween 20 0.1%), 3 times.

 

7. Incubation with secondary antibody (1:5000) at room temperature 1 hr.

 

8.  Wash 10 min with TBST (tween 20 0.1%), 3 times.

 

9. ECL

 

Thanks a lot. 




#170570 Problems of western signal

Posted by cell 293 on 01 September 2014 - 09:11 PM in SDS-PAGE and Western Blotting

Hi everyone

 

I have problem to improve my weak signal.

 

I expect to gel the signal at 35, 50, and 103 kDa. In my data, i have to expose and get the signal around 10~15 min. But the signal is still unclear.

 

What can i do to improve it.

 

In this data, background is every high, but the signals what i want are weak.

 

1.jpg

 

 

In this data. How to get stronger signal? And how to removal the black point in the film?

 

2.jpg  

 

 

This is beta-actin. Sometimes i got two signals when numerous sample was loaded. I am sure i didn't move the film when it was exposed.

 

Is it normal? How to imporve it?

 

3.jpg

 

 

This is simple protocol of my western.

 

1. Dissolve cells by adding PBS and 2X sample buffer  and boiled directly.   (I don't lysis because protein will be lose.)

 

2. Start the electrophoresis process for the sample at 60 V.

 

3. Thansfer proteins at 300 mA, 2 hr.

 

4. Block membrane with 5% non-fat milk in TBS

 

5. Incubation with primary antibody (goat, 1:500) at 4℃ overnight (around 16 hr)

 

6. Wash 10 min with TBST (tween 20 0.1%), 3 times.

 

7. Incubation with secondary antibody (1:5000) at room temperature 1 hr.

 

8.  Wash 10 min with TBST (tween 20 0.1%), 3 times.

 

9. ECL

 

Thanks a lot. 




#166777 metabolic toxicity for non-attached cells

Posted by cell 293 on 08 April 2014 - 01:27 AM in Cell Biology

Hi

 

I want to test the metabolic toxicity for renal cell.

 

I have searched the protocol for how to use S9 fraction.

 

In the protocol. After i treat drug with S9 fot 3~4 hrs, I should to aspirate it off and wash with PBS.

 

But the renal cell that i used is HEK293, it is weak adhesion on well-plate.

 

How can i do to prevent to wash down cells?

 

Thanks




#165700 cell culture and biopsy

Posted by cell 293 on 03 March 2014 - 07:29 PM in Apoptosis, Necrosis and Autophagy

Hi

 

Are apoptosis and nercrosis the same meaning between in vitro and in vivo?

 

Someone told me that necroisis in vitro is different to in vivo, but i don't know what is difference.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.