[someonenew]

hi guys,

i just wanted to know the connection between immunoglobulin and sds-page. i know how sds-page works but i just wanted to know how and why is it used with immunoglobulin.

thanks

[Arkcon]

Lets work together to try and figure this one out -- that's part of the forum rules, doing as much of the work yourself.  Give us a brief definition of what SDS-PAGE is, and what it does, and the structure of immunoglobulins.  And see what sort of answer you can come up with. 

This isn't a very good question however, how and why SDS-PAGE is used on immunoglobulin is like asking, "Why is a pH meter used on a buffer."  There's no how and why, and knowing what a pH meter is and a buffer is doesn't describe some mystical connection between the two.

[Babcock_Hall]

I'll provide a hint of sorts.  Depending on one's choice of conditions for SDS PAGE, one should see different results.

[시1발버러지새끼들아]

I think that your book has an answer...

[Babcock_Hall]

@OP, Do you know what information is generally available in SDS PAGE?  Do you know anything about reducing agents, such as 2-mercaptoethanol?

[someonenew]

sorry for the late reply everyone.... My question really was why is that sds-page electrophoresis the best choice for immunoglobin cleavage products... are there any alternatives??

 I understand the process of which pepsin fragments the immunoglobulin molecule and i get how as the mass of the fragments get heavier the higher up the position on the page it will be.But why do we use this process of analysis?

Also i have another question  I need to understand how to draw a calibration curve. I have a table in excel labelled Molecular Weight (kDa), Log MW and Migration Distance (cm).    but on my page the band pretty much stay the same. it starts off at about 70 kDa and then after the band that represents the 5 minutes it is about 50kDa till the end  (60 minutes). does this mean that the pepsin has cleaved the immunoglobulin moleucle straight away and has stopped after 5 minutes?

I have attached the plate image

[Babcock_Hall]

I see three bands of interest, not two.  Also, it seems to me that the band that is running essentially with the tracking dye is getting more intense with time.  I think that there may be two cleavages.  Is 2-mercaptoethanol present or absent in this experiment?

[someonenew]

no we didnt use that. Basically it was was pepin IgG,citrate buffer and we incubated atdifferent time intervals. 0 1,5,10,20,30 and 60minutes

[Babcock_Hall]

Did you successfully generate a standard curve?  If so, what is the estimated MW of the largest band of the sample.