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Protein chip wash solutions

Protein chip wash solutions - Basic Lab Protocols and Techniques

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  #1  
Old 09-01-2006, 05:33 AM
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Post Protein chip wash solutions



I am a graduate student.
Now i face this problm which i cannot wash protein chip clear, the background of the portein chip have high signals.you know the singals would disturb to get signals from protein spot.
Now the wash solutin include tris-HCL and tween-20, using amido slide.If someone know the wash solutions ingredients,pls tell me. Waiting for your help.Thanks a lot.
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  #2  
Old 09-05-2006, 10:06 AM
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Default Re: Protein chip wash solutions

Hey Jhon,

welcome to the forum!

sorry for the late reply I guess most people were on vacation or the long weekend...

I will check up on this asap.

check for my answer on protein chip washes soon
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Old 09-05-2006, 06:52 PM
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Default Re: Protein chip wash solutions

Most protocols simply wash with TBST or water...

for example:
A Nonredundant Human Protein Chip for Antibody Screening and Serum Profiling


Lueking et al., used TBST to wash during detection and water to wash during protein chip generation.

Generation of Protein Chips—
A microscope slide treated with Bind-Silane (Amersham Pharmacia Biotech, Piscataway, NJ) and a covering slide treated with Repel-Silane (Amersham Pharmacia Biotech) were arranged together, separated by a 30-µm thick metal spacer. The space between the slides was filled with solution of 8% (w/v) polyacrylamide-bisacrylamide (30:0.8). After polymerization, the slides were washed with water and dried. The microscope slides were placed in a Q-Array System (Genetix, New Milton, UK), equipped with humidity control (50%) and 16 blunt-ended stainless steel print tips with a tip diameter of 150 µm. A 13 x 13 spot pattern with a center-to-center distance of 420 µm was printed onto the slides as duplicates. Each spot was loaded five times, resulting in a total transfer volume of 10 nl. These ready-to-use slides can be used without loss of signal giving reproducible results for at least 4 weeks when stored at 4 °C.
Detection Procedure—
After spotting, the protein chips were blocked in 2% (w/v) bovine serum albumin/Tris-buffered saline, 0.1% (v/v) Tween 20 (TBST)1 at room temperature and incubate with primary antibody (mouse-anti-RGSH6 (Qiagen, Valencia, CA) 1:2000 dilution; mouse-anti-GAPDH (Research Diagnostics, Inc., Flanders, NJ) clone 6C5, 1:5,000 dilution; mouse-anti-HSP90ß (Transduction Laboratories, Lexington, KY) clone 68, 1:2,000 dilution), followed by two 10-min TBST washes and incubation with the secondary antibody (rabbit-anti-mouse-IgG-Cye3 (Dianova, Hamburg, Germany), 1:800 dilution) in 2% (v/v) bovine serum albumin/TBST. Subsequently, the protein chips were washed three times for 10 min in TBST-T (0.5% (v/v) Triton X-100) followed by a 5-min wash with Tris-buffered saline. Detection of the signals obtained on these protein microarrays was performed using a 428TM Arrayscanner System (Affimetrix, Palo Alto, CA). Protein microarrays used for serum profiling were blocked in 5% (w/v) fish gelatin/TBST at room temperature, and the serum was added (diluted 1:20 in 5% (w/v) fish gelatin/TBST). After two 10-min TBST washes and subsequent incubation with the secondary antibody (mouse-anti-human immunoglobulin G (IgG; Sigma, St. Louis, MO) 1:5,000 dilution) in 5% (w/v) fish gelatin/TBST, the protein chips were washed three times for 10 min in TBST. This was followed by incubation with the tertiary antibody (rabbit-anti-mouse-IgG-Cye3 (Dianova) 1:800 dilution) in 5% (w/v) fish gelatin/TBST. Subsequently, the arrays were washed three times, each in TBST-T for 20 min. Detection was performed as previously described. All incubation steps were performed for 1 h. All antibody dilutions were in blocking buffer unless otherwise stated.

There are a few commercial sources of protein microarray chip wash buffers.

These include:
Protein Array Wash Buffer
http://www.arraying.com/Products/Buffers_Reagents.html



Other wash buffers that I encountered for protein arrays include:
http://sysbio.harvard.edu/csb/macbea...croarrays.html

PBS
and
PBST (PBS supplemented with 0.1% Tween-20).

For Kinase Chips:

Wash Buffer (WB; 20 mM Tris, 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, pH 7.5).

Kinase Buffer (KB; 50 mM Tris, 10 mM MgCl2, 1 mM DTT, pH 7.5), incubated for 10 min with KB supplemented with 100 µM ATP, and washed for an additional 10 min with KB.


Just a general tip however or a small warning. Make sure the buffers you are using are compatible with your protein chip that you are using!


Check your protein chip manufacturer's manual for further directions and instructions.

Cheers

Last edited by admin; 09-05-2006 at 06:58 PM.
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Old 09-11-2006, 02:52 AM
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Default Re: Protein chip wash solutions

Hello admin.
Thank you much for giving more information about protein chip.I will try your method and improve.I found out that many methods use spin and dry with air brush(CO2) slides after washing slides.Would you tell me you use this method before get signals form slide with CCD scaner?
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Old 09-13-2006, 06:52 AM
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Default Re: Protein chip wash solutions

Thats a good idea to wash with an air brush. How good is it at removing liquids and washing? I think C02 should be the last step, and using a liquid to wash I believe is important.
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Old 09-14-2006, 07:44 AM
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Default Re: Protein chip wash solutions

>>>>>>>
Hello kiki06
I agree with you.spining to remove liquids,But now i cannot find out the spin brack,i can not imagine it.Could you give me more information or pictures?Do you have good idea,pls share with you?
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Old 09-18-2006, 03:11 AM
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Default Re: Protein chip wash solutions

I dont have any pictures, although I think you should read the protocol / manual that came with your protein chip.
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