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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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no MW?
Posted by: Saira (IP Hidden, New member, 2)
Date: February 10, 2005 12:59PM
Hi...I've encountered a problem when I immunostain a membrane with a particular antibody, and the Molecular Weight marker does not show at all when I expose the film. Everything transfers well, including the marker. On some occasions the molecular weight marker shows only faintly. If anybody has any suggestions, I would appreciate it! thanks,
Saira Edited 1 times. Last edit at 02/10/05 01:00PM by Saira.
Re: no MW?
Posted by: nbertos (IP Hidden, New member, 1)
Date: February 11, 2005 01:12PM
Hi Saira;
Forgive me if I misunderstand what you're doing, but as far as I can tell, you're doing western blots? Some antibodies crossreact sufficiently with the MW marker bands to show up on the film, but many (most) do not. Use glow-in-the-dark labels on the plastic covering your membrane so that you can align the film with the membrane after you develop it, and mark the MW marker positions on the film with a permanent marker. Remember not to remove the membrane from the plastic cover with the label before aligning all your films! Hope this helps, Nick
Re: no MW?
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 15, 2005 03:31AM
Hi it seems you are using a Chemiluminescent substrate for your Western. in order to have your markers on your X-ray fim use the Chemiluminescent BlueRanger Marker Mix from Pierce, this will serve your purpose effeciently.
[www.piercenet.com] Good Luck
home made MK
Posted by: JohnH (IP Hidden, Unregistered user, )
Date: March 15, 2005 05:06AM
hi,
Is there any way to home-make immunoblot chemiluminescent MK? Anyone can give a working protocol? Thax.
Re: home made MK
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 15, 2005 05:36PM
Hi,
Although all of these markers are only available commercialy, hwoever, one can assume that they may have constructed these proteins with an HRP which is acounted for in their molecualr weight, or they have biotin labeled these proteins in the markers and have coupled them individually to Strep avidin-HRP whic is also acounted for in the molecualr weight of each protein and thus you get at the end a Chemiluminescent protein mix and it will only appear when you add the Chemiluminescent substrate at the last step of your Western blot procedure. Another altrantive is to have mix of His-tag fusion proteins with known molecular weight as your protein marker (from niebering labs), however you need to incubate your Western blot with your primary antibody and anti-His- tag protein to reveal your protein and the protein marker (His-tag proteins mix). I hope this information is helful for you, good Luck!
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