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Photoaffinity labeling

Photoaffinity labeling - Protocols and Methods Forum

Photoaffinity labeling - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-21-2003, 07:21 AM
jmeyer1
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Default Photoaffinity labeling



Hello,

I want to start an photoaffinity labeling experiment, unfortunately I
have no idea which light source will be sufficient for this purpose. I
want to use an Azidogroup (254nm)and a Benzoylgroup (366nm) as
photoactivateable structure. I compared the light sources given in
serveral papers. According th them everything from a 500W Xenon to a TLC
UV-cabinet can be used. The iinformation given is not very detailed
(Numeber ofWatts is missung etc,) so I am not to sure about that. Has
anyone any ideas/experience (...supplier)?
Any hints will be greatly appreciated,

thanx Jens

PS The light source should be as inexpensive as possible (...as always
when working at the university )

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  #2  
Old 10-31-2003, 05:48 AM
Dr Engelbert Buxbaum
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Default Photoaffinity labeling

jmeyer1 @uni.bonn.de> wrote:



It appears that the best results are obtained with high power flash
bulbs (1000 W and more). These can do the crosslinking fast. Low power
lamps require much more time, you get problems with sample heating,
evaporation and what have you. So its probably best to find somebody
with appropriate equipment and enter cooperation.

On the other hand, I have done croslinking of ATP to an ATPase with
nothing more than the sterilising light in a class II cabinet, the
sample was in a thin layer and kept on ice, with 10 min illumination
time giving maximum signal.
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  #3  
Old 10-31-2003, 12:17 PM
Han
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Default Photoaffinity labeling

Dr Engelbert Buxbaum <[Only registered users see links. ]> wrote in news:bnst3i
$k2t$04$[Only registered users see links. ]:


Would I be able to find details on youyr technique in this article?

Co-operative binding sites for transported substrates in the multiple
drug resistance transporter Mdr1.
Eur J Biochem. 1999 Oct 1;265(1):64-70.

Thanks in advance!
--
Best regards
Han
email address is invalid.
Please use "mj" nothing "broek" at "med" dot "cornell" dot "edu"
Sorry for the munging
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  #4  
Old 11-04-2003, 05:34 AM
Dr Engelbert Buxbaum
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Default Photoaffinity labeling

Han wrote:



Nope, I published that experiment in Biochem. J. 318 (1996) 923-929. But
there is realy not much to it, I used a watch glass placed on ice, and
put the whole thing as closely as possible to the lamp. Note that with
ATP you do not need a photoactivatable group, it crosslinks all by it
self. You should take samples every min or so to determine the time
required for maximum signal.
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  #5  
Old 11-04-2003, 12:06 PM
Han
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Default Photoaffinity labeling

Dr Engelbert Buxbaum <[Only registered users see links. ]> wrote in
news:bo7dpr$q5r$05$[Only registered users see links. ]:


Thanks Engelbert!

--
Best regards
Han
email address is invalid
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