Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

starry sky western blot... lots of spots.


  • Please log in to reply
5 replies to this topic

#1 MaggieRoara

MaggieRoara

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 09 March 2009 - 02:11 AM

My western blot has alot of spots on it. If secondary antibody incubation is for 1hr, i dont see any bands but i see spot, somthineg like a starry sky. When i incubate secondayr Ab for 1hr 30 min, i see some bands, but with starry sky as well. i am thinking this might be because my secondary Ab is of too high concentration (1:1000). Could it be because the high concetration means the secondary Abs are conjugating with each other? I am trying with a lower concetration (1"10,000). I'll post back how that looks, but please help?

#2 WHR

WHR

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 49 posts
0
Neutral

Posted 09 March 2009 - 04:31 AM

My western blot has alot of spots on it. If secondary antibody incubation is for 1hr, i dont see any bands but i see spot, somthineg like a starry sky. When i incubate secondayr Ab for 1hr 30 min, i see some bands, but with starry sky as well. i am thinking this might be because my secondary Ab is of too high concentration (1:1000). Could it be because the high concetration means the secondary Abs are conjugating with each other? I am trying with a lower concetration (1"10,000). I'll post back how that looks, but please help?



hi,
Your antibody may have aggregates in it.
You may centrifuge the antibody at top speed for 5 minutes to remove the aggregates.

#3 jah

jah

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 09 March 2009 - 05:11 AM

In addition to WHR's comment ,here's my quick trouble shooting list for starry sky blots:
If using PVDF, did you pre-wet the membrane with MeOH before equilibrating in transfer buffer?
Did you squeeze all of the bubbles from between the gel and the membrane during the transfer?
If you use alcohol in your transfer buffer, did you wash the membrane to remove the alcohol? Residual MeOH can cause the blocking protein to aggregate on the membrane, and cause spots.
What are you using to block? Sometimes if your blocking protein, especially dry milk, isn't fully dissolved, you can get the 'starry sky' effect.
Assuming you're using chemiluminescence, and Saran wrap between the membrane and film...bubbles may again be the culprit.

Edited by jah, 09 March 2009 - 05:11 AM.


#4 mikew

mikew

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 80 posts
0
Neutral

Posted 09 March 2009 - 11:09 AM

Hi,

Starry sky can come from several sources.
1. Too high concentration of secondary. Use 1:3000.
PS. What isotype is your secondary? Usually starry sky is associated with a rabbit.
2. The Sponge pads you are using for transfer are contaminated with protein. This leads to protein going from the sponges onto your blot. Wash sponges thoroughly.
Also, use 2 Watmann filters on each side of the transfer. This helps.

3. This may be the MOST important. What type of Skim Milk are you using as a blocking reagent?
Sounds funny but this is no joke. We've gotten starry sky Westerns (only with rabbit secondary) when we buy cheap skim milk powder. Carnation brand works but I highly recommend APEX non-fat skim milk from Genesee scientific. It cost $18.00 per 500 grams. We have had NO starry sky when using this.

Good luck.

#5 mikew

mikew

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 80 posts
0
Neutral

Posted 09 March 2009 - 11:22 AM

One more thing about the milk.
When making the milk in TBST stir it with a stir bar for quite some time.
This will help dissolve particulate matter that can stick to your blot.

#6 MDavies

MDavies

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 13 October 2009 - 09:43 AM

One more thing about the milk.
When making the milk in TBST stir it with a stir bar for quite some time.
This will help dissolve particulate matter that can stick to your blot.


When the milk looks dissolved, I send the milk through a 200 um filter -- actually not a filter, a "cell strainer" -- to get rid of aggregates that won't dissolve. Is that sufficient?




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.