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wdyeo's Content

There have been 41 items by wdyeo (Search limited from 18-July 18)



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#161481 10x Laemmli buffer

Posted by wdyeo on 21 October 2013 - 04:36 AM in SDS-PAGE and Western Blotting

sorry for the late reply ~




#161480 10x Laemmli buffer

Posted by wdyeo on 21 October 2013 - 04:35 AM in SDS-PAGE and Western Blotting

Hi, 

Yeah...maybe...

I hope at least can fix 20ul...

Hopefully my lab has the big gel setting...:D
Have a nice day ~




#161220 10x Laemmli buffer

Posted by wdyeo on 14 October 2013 - 02:57 AM in SDS-PAGE and Western Blotting

Hi doxoburicin, 

Thanks alot for the advice and i really agree with you

 

But i just found this, to make 10X stock here! 

U think it will work?

 

http://openwetware.o...is_formulation)

 

Variant

To make 10 ml of 10x stock

  • In 70 % glycerol / 30 % water, dissolve the following:
    • 0.606 g Tris-base
    • 2.5 g SDS
  • Adjust the pH using pH indicator strips to 6.8
  • Add 2 mg of Bromophenol Blue and make sure the powder is completely dissolved
  • Adjust the final volume to 10 ml with 70 % glycerol / 30 % water before storing at -20°C.
  • Add the appropriate volume of a β-mercaptoethanol 100% stock to your samples just before denaturing them at 95°C.

 

 

Thank you.




#161173 10x Laemmli buffer

Posted by wdyeo on 12 October 2013 - 09:27 AM in SDS-PAGE and Western Blotting

Dear all, 

Is there any recipe for home-made 10x Laemmli buffer?

My sample is just too low concentration but the common stock is 2x,4x,5x and 6x...

And is it possible not to use powder form of SDS? I don't know how to dissolve it and we have 20% SDS in solution.

Thank you.




#161082 Semi-dry system for NuPage with MES buffer

Posted by wdyeo on 10 October 2013 - 02:18 AM in SDS-PAGE and Western Blotting

Hi mdfenko, 

Thanks alot for the advices and is really nice of you to share the handbook and link.

I will try again.

Have a nice day :D




#160948 Semi-dry system for NuPage with MES buffer

Posted by wdyeo on 06 October 2013 - 03:36 AM in SDS-PAGE and Western Blotting

Hi mdfenko, 

 

Thanks alot for the reply.

 

I am using a 20% methanol + 0.02% of SDS in my transfer buffer, which is a 1X tris and glycine.




#160827 Semi-dry system for NuPage with MES buffer

Posted by wdyeo on 01 October 2013 - 01:26 PM in SDS-PAGE and Western Blotting

Dear all, 

 

I am using a 4-12% Bis-Tris NuPage (Invitrogen) ready-made gel , and run in MES buffer to enable me to view the protein size of less than 10k Da.

However, when i transferred the gel to the Immobilon PVDF 0.2um using Trans-Blot SD Semi-Dry (Biorad), at 12V for 45 min for the mini gel , the transferring is still incomplete when i stained my gel with coomasie blue after transferring it.

 

The transfer buffer which I am using is theglyicine , tris base, SDS and methanol.

I did not use the running buffer as suggested for NuPage transfer buffer.

Is it because of the different transfer buffer which causes the inefficiency of the transferring?

Thank you.

 




#160497 Amount of EDTA

Posted by wdyeo on 21 September 2013 - 03:30 AM in SDS-PAGE and Western Blotting

Ok.

Thanks alot :D




#160384 Amount of EDTA

Posted by wdyeo on 19 September 2013 - 02:08 AM in SDS-PAGE and Western Blotting

Hi, 

Suppose i use 1mM of EDTA for 500 mL of 20x MES buffer.

But i use 1 mM of EDTA for 250 mL of 20x MES buffer.

Will it affects? :(




#160376 Amount of EDTA

Posted by wdyeo on 18 September 2013 - 01:57 PM in SDS-PAGE and Western Blotting

Dear all, 

I made a silly big mistake by adding twice more the amount of EDTA for the MES buffer...

Will it has much affect on it?

I do not have MES and other chemicals to add to make the amount equivalent for the extra EDTA :(

 




#159131 Common causes for low RNA A260/230 ratios

Posted by wdyeo on 19 August 2013 - 01:36 PM in PCR, RT-PCR and Real-Time PCR

Hi Memari, 

Thanks alot for the reply ~!

Have a nice day biggrin.png




#158558 Common causes for low RNA A260/230 ratios

Posted by wdyeo on 04 August 2013 - 11:49 AM in PCR, RT-PCR and Real-Time PCR

Hi robradford,

 

I am interested to know whether you put all the isolated RNA (after extraction from Trizol) to the Qiagen RNeasy Mini, by adding the RLT buffer and the subsequent step for the clean up?

I had extracted RNA from Trizol but my ration 260/230 is too low and I am wondering what shall i do to improve the ratio?

Thank you~ :D




#158127 Protein extraction from mice/rat pancreas

Posted by wdyeo on 23 July 2013 - 02:42 PM in SDS-PAGE and Western Blotting

Dear all,
May i know the best method for protein extraction from mice/rat pancreas?
Thank you~



#157049 Selection of needle size to homogenize cells for RNA extraction

Posted by wdyeo on 28 June 2013 - 02:45 AM in PCR, RT-PCR and Real-Time PCR

If you do not use syringe and needle, how do you homogenize your cells?
Thanks ~



#156953 Selection of needle size to homogenize cells for RNA extraction

Posted by wdyeo on 26 June 2013 - 02:59 AM in PCR, RT-PCR and Real-Time PCR

Dear All,
I used RNeasy Mini Qiagen to extract RNA from cell culture.
But if i use 26G needle instead of 20G needle (as written down in the handbook) to homogenize the cells in cell culture, will it affects the quality of RNA or i loss more RNA etc?
Help..


Edited to make font readable. Bob



#156952 Less than 10 GFP positive cells

Posted by wdyeo on 26 June 2013 - 02:56 AM in Flow Cytometry

Hi,
Thank you for the suggestion.
Have a nice day :D



#156008 Sensitivity of RT-PCR and qPCR

Posted by wdyeo on 04 June 2013 - 12:37 AM in PCR, RT-PCR and Real-Time PCR

Oh...
okok..thanks alot ya ! :D



#155765 Sensitivity of RT-PCR and qPCR

Posted by wdyeo on 29 May 2013 - 08:45 AM in PCR, RT-PCR and Real-Time PCR

Hi,
Thanks alot for the suggestion but I am confuse, how do i take the harvested tissues and spike them on the normal tissues/cells?
Thank you.



#155589 Less than 10 GFP positive cells

Posted by wdyeo on 26 May 2013 - 01:55 PM in Flow Cytometry

Dear All,
I harvested the tissues (such as pancreas, liver and spleen) in order to detect if there is any GFP+ cells
Is it possible to analyse/sort the tissues samples using FACS to quantify the number of GFP+ cells which might be less than 10 cells!?!
Thank you.



#155588 Sensitivity of RT-PCR and qPCR

Posted by wdyeo on 26 May 2013 - 01:50 PM in PCR, RT-PCR and Real-Time PCR

Dear All,
I inject GFP stem cells into a normal mouse and i harvested the tissues (such as pancreas, liver and spleen) in order to detect if the GFP+ cells are successfully transplanted/home into the mouse.
Is it possible for me to do RT-PCR or qPCR to detect the level of GFP+ cells? Which is more sensitive because maybe only 1 or 2 GFP+cells in whole tissues..Or any other technique besides immunohistochemistry?
I have no idea if the GFP+ cells are transplanted successfully into the mouse by the way :P
Thank you.



#154888 restain secondary antibody

Posted by wdyeo on 10 May 2013 - 12:58 AM in Histology and Pathology

HI,
OK, Thanks alot for the suggestion :D



#154830 restain secondary antibody

Posted by wdyeo on 09 May 2013 - 12:24 AM in Histology and Pathology

Yeah....but i do not have the sample already :<
May i know what is the commonly used stripping protocols...at least there's still some slight hope to try...
Thank you.



#154544 restain secondary antibody

Posted by wdyeo on 03 May 2013 - 06:18 AM in Histology and Pathology

Hi,
The problem i am facing now is, i try to identify GFP staining in my tissues and one of the primary antibody is against the GFP and i am supposed to stain the GFP with secondary in green to enhance the GFP signalling but i made a silly mistake, using red for the GFP antibody Posted Image .



#154522 restain secondary antibody

Posted by wdyeo on 02 May 2013 - 12:37 PM in Histology and Pathology

Dear all,

I am doing the immunohistochemistry on the tissues, in the gel about 150um (cut with vibratome).
I made a mistake in my secondary antibody, instead of using secondary anti-rabbit green + secondary anti-mouse red; i used secondary anti-rabbit red and secondary anti-mouse green . I did a double staining for my primary antibodies, one is against rabbit and the other is mouse.

Is there a way to remove the secondary antibody and restain my tissues slice?
Thank you.



#154157 Oil red o - IF

Posted by wdyeo on 24 April 2013 - 02:59 AM in Tissue and Cell Culture

Hi bob1,
Thanks alot for the point.
Have a nice day :D




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