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Wester blot analysis


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#1 hidayahcellculture

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Posted 27 November 2013 - 09:57 PM

Hi all molecular biologists, 

 

I will do western blot analysis.

but i still confuse in selecting the primary and secondary antibodies.

 

my sample is human fetal osteoblastic cell lines. I need to check for some protein.

 

for primary antibody, i would like to use anti osteocalcin , anti AP and anti integrin beta3.

 

my Questions:

1. how i suppose to write the primary antibody to order from company?

2. what are the secondary antibody i need to use?

 

thank you for helping me  smile.png  

 

 



#2 hidayahcellculture

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Posted 27 November 2013 - 10:07 PM

Hi all . good day. 

 

I will do western blot analysis.

but i still confuse in selecting the primary and secondary antibodies.

 

my sample is human fetal osteoblastic cell lines. I need to check for some protein.

 

for primary antibody, i would like to use anti osteocalcin , anti AP and anti integrin beta3.

 

my Questions:

1. how i suppose to write the primary antibody to order from company?

2. what are the secondary antibody i need to use?

 

thank you for helping me  smile.png



#3 bob1

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Posted 28 November 2013 - 12:11 AM

Your best bet is to look through the literature and find out which antibodies people use, and where they got them from.  Often you will find that there will be one or two different primary antibodies for the same protein that everyone uses.  Then just send an order to that company.

 

The secondaries you need to use depend on the species the primary antibodies were raised in.  In general there are 3 species people use for generating antibodies, mouse, rabbit and goat.  The secondaries will say something like "goat anti-mouse IgG" or "Donkey anti-rabbit IgG".  You need one for each different species the primary antibodies were raised in.  Make sure that you get ones coupled to the appropriate chemistry for your application.  For western blotting this is commonly Horse radish peroxidase (HRP), or alkaline phosphatase (AP) conjugated.

 

Dako is a reliable, but expensive company for all things antibody related.



#4 hidayahcellculture

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Posted 28 November 2013 - 01:31 AM

dear moderators, thank you for the info.

 

i would like to ask more questions:

 

 why it is important to know where the antibody comes from?

 

and if i use the primary antibody (example:source from goat),is that compulsory to use the second antibody from the same source?

 

and what are the effects of using antibody from source: goat/rabbit/horse et towards my sample (human fetal osteoblast cell line)?

 

what are the precautions i should consider  in selecting the primary and secondary antibodies to detect proteins of interest  in my sample? 



#5 2xzwei

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Posted 28 November 2013 - 04:07 AM

Apparently you know what proteins you're looking for, right? So just browse vendor products online and order what you need (order number and amount should be enough).

 

The primary antibody should give the information in which host it was produced. So if you find anti-osteocalcin AB that was raised in goat you have to order a secondary anti-goat (-HRP linked) antibody for detection. If your primary anti-CD61 was made in rat - you order anti-rat (-HRP).

 

I would recommend you to first check publications and/or manufacturer's references that can validate that the ordered primary AB is working and gives good results...



#6 bob1

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Posted 28 November 2013 - 12:36 PM

If you don't know the species of the primary, you can't probe with a secondary - as that needs to match the primary. 

 

If you have a goat primary you MUST NOT use a secondary raised in a goat, you need a secondary antibody raised against goat IgG, which will be done in another species, usually donkey or horse.

 

There are no problems using antibodies of different sources, so long as they were raised against the human protein. 

 

Precautions - some antibodies are "dirty" (give high background or non-specific binding), usually the cleanest antibodies are mouse, then rabbit may be a bit dirtier, then goat, which is often dirty, but usually fine.  All antibodies need to be optimised in terms of working dilution and  conditions for use.  Be aware that some antibodies will not work on western blots (denatured protein) as they are raised against undenatured proteins.  Always check the datasheet before purchasing, it should say something like "validated for western blot".






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