Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Histology Forum > Immunohistochemistry Forum
Register Search Today's Posts Mark Forums Read

Immunohistochemistry Forum Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections.


non specific binding of FITC to erythrocytes

non specific binding of FITC to erythrocytes - Immunohistochemistry Forum

non specific binding of FITC to erythrocytes - Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-12-2009, 01:02 PM
Pipette Filler
Points: 91, Level: 1 Points: 91, Level: 1 Points: 91, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Smile non specific binding of FITC to erythrocytes



Hi,

Im working with FISH on paraformaldehyde-fixed cells. The cells are labelled with a DIG-labeled RNA probe, subsequently labeled with an anti-DIG-HRP and then finally detected with FITC that reacts with the HRP. I get high unspecific binding to my erythrocytes without using the probe i.e on control slides where Ive added only the FITC. I know that FITC is negatively charged and will bind positively charged molecules, but does anyone know how I can efficiently block/treat my slides in order to get rid of the unspecific binding ?

Regards,

Elena
Reply With Quote
  #2  
Old 06-15-2009, 02:00 AM
Graduate Student
Points: 2,354, Level: 31 Points: 2,354, Level: 31 Points: 2,354, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 114
Thanks: 19
Thanked 15 Times in 14 Posts
Default Re: non specific binding of FITC to erythrocytes

I am experiencing unspecific binding of my FITC to my streptavidin-coated microspheres too. Basically, I have streptavidin-coated microspheres to which biotinylated DNA and biotinylated anti-HA antibodies bind to. The biotinylated DNA actually has a sequence of HA at the 3' end, so after in vitro translation, peptides with HA tags are produced and they bind to the anti-HA antibodies on the beads. I detect these proteins using a peptide that is conjugated to FITC. During my analysis with flow cytometry, I realized that I get varying signals.

And in my negative control, there is supposed to be no DNA at all so no proteins are produced and no peptide-FITC should bind. But I get fluorescence signals in the negative controls and this indicates unspecific binding.

Been stumped at finding a way to get rid of the unspecific binding, though the only thing for now I can think of is to perform more stringent washings in between each step.
Reply With Quote
Reply

Tags
binding , erythrocytes , fitc , specific


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
how to reduce IgG non specific binding? dizue Antibody Forum 2 12-02-2008 09:42 AM
Calculations for specific activity Morales, Victor Protein Forum 0 10-02-2008 01:17 PM
ELISA for protein-protein binding assay takayama Protein Forum 1 06-27-2006 12:08 PM
Question about binding studies Dr Engelbert Buxbaum Protocols and Methods Forum 0 04-05-2004 09:44 AM
Sequence non-specific DNA binding EK Protocols and Methods Forum 5 08-28-2003 02:41 AM


All times are GMT. The time now is 02:23 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11961 seconds with 16 queries