Bitan:dot blot



       Preparation of A&#946; oligomers



Preparation of A&#946; oligomers

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<ul style='margin-top:0in' type=disc> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list .5in'>Prepare HFIP films for A&#946;.</li>

<li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list .5in'>Dissolve the HFIP film in NaOH so that the concentration of A&#946; is 2mM.</li> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list .5in'>Sonicate for 30sec.</li> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list .5in'>Dilute this solution to 45 &#956;M.</li> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list .5in'>Spin at 14K for 10 min.</li> <li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list .5in'>Take the supernatant and keep it on the bench top.</li>

<li class=MsoNormal style='mso-list:l0 level1 lfo1;tab-stops:list .5in'>Oligomers start to form from the very beginning.</li> </ul>

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<body lang=EN-US link=blue vlink=purple style='tab-interval:.5in'>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> Dot Blot Development</o:p> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Dot Blotting Protocol</o:p></b>

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Apply peptides (different preparations) on defined spots in 2-4uL blots (I used 2uL)

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o

<![endif]>Make sure the amount of peptide from each preparation in each dot is the same in terms of moles

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Let them air dry

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Membrane Development</o:p></b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>**** <span class=GramE>washes use 0.1% Tween ; with antibodies use 0.05% Tween (by volume)</o:p></b>

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Dip membrane in appropriate blocking solution (5-6% skim milk) in TBS- Tween  <span class=GramE> ( 0.05%) (<span class=SpellE>tris buffered saline) for 30 min

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o

<![endif]>Since <b>milk contains a generic mix of a relatively high concentration of known proteins</b>, 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.

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo2'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'> § <![endif]><a href="http://wiki.answers.com/Q/Why_you_use_skim_milk_to_block_the_membrane_in_western_blotting">http://wiki.answers.com/Q/Why_you_use_skim_milk_to_block_the_membrane_in_western_blotting</a>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o <![endif]>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.

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo2'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'> § <![endif]><a href="http://www.protocol-online.org/biology-forums/posts/37795.html">http://www.protocol-online.org/biology-forums/posts/37795.html</a>

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o <![endif]>Dilute primary antibody in blocking solution and keep at room temperature for 2 hours or overnight at 4 degrees C

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o <![endif]>Use 2 membranes (For standardization; you may have to use more)

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo2'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'> § <![endif]>A1: use 1:5000 dilution (1 uL in 5mL = 2uL in 10mL)

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.5in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level3 lfo2'><![if !supportLists]><span style='font-family:Wingdings;mso-fareast-font-family:Wingdings;mso-bidi-font-family: Wingdings'> § <![endif]>A2: use 1: 10000 dilution (1uL in 10mL)

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Wash membrane 3X in TBS- Tween

for 10 min each

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Wash membrane in TBS once for 10 min

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Block in blocking solution (5% skim milk in TBST) for 30min

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Dilute secondary antibody in blocking solution and incubate for 1 hour at room temperature

<p class=MsoListParagraphCxSpMiddle style='margin-left:1.0in;mso-add-space: auto;text-indent:-.25in;mso-list:l1 level2 lfo2'><![if !supportLists]><span style='font-family:"Courier New";mso-fareast-font-family:"Courier New"'><span style='mso-list:Ignore'>o <![endif]>Use 1:10000 dilution (Standardize for your antibody)

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Wash 3 times with TBS- Tween for 10min

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Wash once with TBS for 10 min

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Detection</o:p></b>

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Mix detection solution (1.5mL Solution A: 1.5mL solution B)

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Cover membrane with solution in dark for 10 min

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Soak up excess detection solution with large <span class=SpellE>kimwipe folded over many times

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Develop blots using ECL sensitive camera (Spencer lab)

<p class=MsoListParagraphCxSpLast> </o:p>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Reagents:</o:p></b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>TBS – <span class=SpellE>Tris Buffered Saline</o:p></b>

<p class=MsoNormal>25mM Tris, 150mM <span class=SpellE>NaCl, 2mM KCl , pH 8

<span style='font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif"; mso-fareast-font-family:Calibri;mso-bidi-font-family:"Times New Roman"; mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'><br clear=all style='page-break-before:auto;mso-break-type:section-break'>

<p class=MsoNormal>1X TBS

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>8g NaCl

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>0.2g KCl

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>3g Tris Base

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>800mL ddH2O

<p class=MsoNormal>10X TBS

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>80g NaCl

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'>

<![endif]>2g KCl

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>30g Tris base

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>800mL ddH2O

<span style='font-size:11.0pt;line-height:115%;font-family:"Calibri","sans-serif"; mso-fareast-font-family:Calibri;mso-bidi-font-family:"Times New Roman"; mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA'><br clear=all style='page-break-before:auto;mso-break-type:section-break'>

<p class=MsoNormal>Directions

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Dissolve all dry reagents together in 800mL of ddH2O

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'>

<![endif]>Adjust pH to 8

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Add ddH2O to final volume of 1L

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Sterilize by autoclaving (probably don’t need this for my experiments

<p class=MsoNormal>From http://www.cytographica.com/lab/solutions/TBS.htm

<p class=MsoNormal> </o:p>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>TBS-<span class=SpellE>Tween (500mL)</o:p></b>

<p class=MsoNormal>0.1% Tween-20 in 1X Tris Buffered saline – pH 8

<p class=MsoNormal>Reagents

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'>

<![endif]>2.5mL 20% Tween 20

<p class=MsoListParagraphCxSpMiddle style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>50mL 10X TBS

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>447.5mL ddH2O

<p class=MsoNormal>Directions

<p class=MsoListParagraphCxSpFirst style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Add 50mL of 10X TBS to 447.5 mL ddH2O

<p class=MsoListParagraphCxSpLast style='text-indent:-.25in;mso-list:l1 level1 lfo2'><![if !supportLists]><span style='mso-bidi-font-family:Calibri'> -<span style='font:7.0pt "Times New Roman"'> <![endif]>Add 2.5mL of 20% Tween-20 and mix

<p class=MsoNormal>From http://www.cytographica.com/lab/solutions/TBST.htm

<p class=MsoNormal> </o:p>

<p class=MsoNormal> </o:p>