Bitan:dot blot

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Preparation of Aβ oligomers

Preparation of Aβ oligomers

 

 

  • Prepare HFIP films for Aβ.
  • Dissolve the HFIP film in NaOH so that the concentration of Aβ is 2mM.
  • Sonicate for 30sec.
  • Dilute this solution to 45 μM.
  • Spin at 14K for 10 min.
  • Take the supernatant and keep it on the bench top.
  • Oligomers start to form from the very beginning.

Dot Blot Development

Dot Blotting Protocol

-          Apply peptides (different preparations) on defined spots in 2-4uL blots (I used 2uL)

o   Make sure the amount of peptide from each preparation in each dot is the same in terms of moles

-          Let them air dry

Membrane Development

**** washes use 0.1% Tween; with antibodies use 0.05% Tween (by volume)

-          Dip membrane in appropriate blocking solution (5-6% skim milk) in TBS-Tween (0.05%) (tris buffered saline) for 30 min

o   Since milk contains a generic mix of a relatively high concentration of known proteins, it is used to block the parts of the membrane that don't already have protein on so that the antibodies won't bind there. As it is very cheap and readily available, it is a good source of protein to use. It is unlikely that a Western blot would be needed for any protein that is contained in milk, but if that is the case an alternative mixture can be used.

§  http://wiki.answers.com/Q/Why_you_use_skim_milk_to_block_the_membrane_in_western_blotting

o   5 g non-fat dry milk
100 ml TPS (most people use PBS but both work great)

-dissolve non-fat dry milk in buffer
-adjust pH to 7.4 (or what ever you need it to be)
-In ultra-centrifuge spin at 100,000 g for 1 hr
- collect supernatant and filter through a standard 0.2 micron filter

Store at 4 degree C

Do this and your buffer will be a nice clear liquid but still have a high concentration of Cassein, which is the protein in milk that does the actual blocking... Note also, that this means your buffer is sterile as well. Most people don't bother to filter the buffer after dissolving the milk in it, likely because it's very difficult to do. If you don't use an ultra-centrifuge first the solution is too thick to get through most filters; it absolutly will NOT go through a 0.2 micron filter.

§  http://www.protocol-online.org/biology-forums/posts/37795.html

o   Dilute primary antibody in blocking solution and keep at room temperature for 2 hours or overnight at 4 degrees C

o   Use 2 membranes (For standardization; you may have to use more)

§  A1: use 1:5000 dilution (1 uL in 5mL = 2uL in 10mL)

§  A2: use 1: 10000 dilution (1uL in 10mL)

-          Wash membrane 3X in TBS-Tween for 10 min each

-          Wash membrane in TBS once for 10 min

-          Block in blocking solution (5% skim milk in TBST) for 30min

-          Dilute secondary antibody in blocking solution and incubate for 1 hour at room temperature

o   Use 1:10000 dilution (Standardize for your antibody)

-          Wash 3 times with TBS-Tween for 10min

-          Wash once with TBS for 10 min

Detection

-          Mix detection solution (1.5mL Solution A: 1.5mL solution B)

-          Cover membrane with solution in dark for 10 min

-          Soak up excess detection solution with large kimwipe folded over many times

-          Develop blots using ECL sensitive camera (Spencer lab)

 

Reagents:

TBS – Tris Buffered Saline

25mM Tris, 150mM NaCl, 2mM KCl, pH 8


1X TBS

-          8g NaCl

-          0.2g KCl

-          3g Tris Base

-          800mL ddH2O

10X TBS

-          80g NaCl

-          2g KCl

-          30g Tris base

-          800mL ddH2O


Directions

-          Dissolve all dry reagents together in 800mL of ddH2O

-          Adjust pH to 8

-          Add ddH2O to final volume of 1L

-          Sterilize by autoclaving (probably don’t need this for my experiments

From http://www.cytographica.com/lab/solutions/TBS.htm

 

TBS-Tween (500mL)

0.1% Tween-20 in 1X Tris Buffered saline – pH 8

Reagents

-          2.5mL 20% Tween 20

-          50mL 10X TBS

-          447.5mL ddH2O

Directions

-          Add 50mL of 10X TBS to 447.5 mL ddH2O

-          Add 2.5mL of 20% Tween-20 and mix

From http://www.cytographica.com/lab/solutions/TBST.htm

 

 

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