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Protoarray™ Microarray—Protein-Protein Interaction (PPI) Application

Protoarray™ マイクロアレイ PPI アプリケーションでプローブとして使用したいタンパク質を精製し、目的のタンパク質に対する抗体を用いてウェスタン検出して、シグナルが得られませんでした。 何が起きたのでしょうか？

Here are possible causes and solutions:

Cause

Solution

Poor or incomplete transfer Monitor the transfer of pre-stained protein standard bands to determine the transfer efficiency. 
Insufficient exposure time 

Increase the exposure time. 

Epitope tag not present or cleaved 

-Confirm the presence of the tag by sequence analysis and ensure the tag is cloned in frame. 

-Perform all purification steps at 4 degrees C and use protease inhibitors to prevent proteolytic cleavage of the tag. 

PPI アプリケーション用のプローブとして使用するためにタンパク質をビオチン標識しようとしていますが、ビオチン標識が非常に少ないかまたは全く得られていません。 何が問題ですか？

Here are possible causes and solutions:

Cause

Solution

Incorrect buffers used or the biotinylation reaction is not performed correctly Make sure the protein is in a buffer that does not contain any primary amines such as ammonium ions, Tris, glutathione, imidazole, or glycine. 
Make sure the biotinylation reaction was performed correctly using the specified molar ratios and at pH ~8.0. Check that the calculations and serial dilutions are performed correctly. 
Protein has low lysine content or lysine residues are not readily available for biotinylation 

Perform the biotinylation reaction at a higher molar ratio. You may express your protein as a fusion to a tag that contains lysine. 

PPI アプリケーション用のプローブとして使用するためにタンパク質をビオチン標識しようとしていますが、余分なビオチン標識バンドがみられます。 何が生じているのでしょう？

If protein impurities are present and undergo biotinylation, then high background during probing may result. We recommend purifying the protein to remove impurities before performing the biotinylation reaction. 

Protoarray™ マイクロアレイをタンパク質プローブでプローブして、得られたシグナルは非常に弱いかまたは全くありませんでした。 どのような解決策が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Epitope tag not present or not accessible Confirm the presence of the tag by western detection. Ensure the tag is accessible under native conditions by performing an ELISA. 
Poor biotinylation of protein probe 

Incorrect buffers used or the biotinylation reaction is not performed correctly:

-Make sure the protein is in a buffer that does not contain any primary amines such as ammonium ions, Tris, glutathione, imidazole, or glycine. 

-Make sure the biotinylation reaction was performed correctly using the specified molar ratios and at pH ~8.0. Check that the calculations and serial dilutions are performed correctly.

Protein has low lysine content or lysine residues are not readily available for biotinylation:

- Perform the biotinylation reaction at a higher molar ratio. You may express your protein as a fusion to a tag that contains lysine.

Low probe concentration

Perform probing with higher probe concentration or increase the incubation time. 
Incorrect probing procedure Follow the recommended protocol for probing on page 23 of the manual. Be sure all incubations are performed at 4 degrees C. Prepare the Blocking Buffer and Washing Buffer freshas described on page 19-20 of the manual.
Do not allow the array to dry during the probing procedure. 
Avoid prolonged exposure of detection reagents labeled with a fluorescent dye to light. 
Incorrect scanning or imaging Scan the array at suitable wavelength for the detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source. 
Decrease stringency Decrease the number of washes. Perform probing and washing in the absence or lower concentration of detergent or salts. 
Protoarray™ マイクロアレイをタンパク質プローブでプローブして、非常に高いバックグラウンドが生じました。 どうすればよいでしょうか？

Here are possible causes and solutions:

Cause

Solution

Improper blocking Prepare the Blocking Buffer fresh as described on page 19 of the manual.
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the Washing Buffer fresh as described on page 20 of the manual. 
Array dried during probing Do not allow the array to dry during probing. 
Array not dried properly before scanning Dry the array as described on page 24 of the manual before scanning. 
High probe concentration Decrease the probe concentration or decrease the incubation time. 
Antibody cross-reactivity Probe a protein array using only the antibody without the protein probe to detect cross-reactivity with the antibody only. 
Protoarray™ マイクロアレイをタンパク質プローブでプローブしたところ、非常に不均一なバックグラウンドが得られました。 何かヒントはありますか？

Here are possible causes and solutions:

Cause

Solution

Uneven blocking or washing During the blocking or washing steps, ensure the array is completely immersed in blocking solution or Washing Buffer, and use at least 5 mL buffer in the incubation tray to cover the array completely with buffer. 
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the Washing Buffer fresh as described on page 20 of the manual. 
Portions of array have dried Do not allow the array to dry during probing. 
Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps. Take care while inserting the array into the incubation tray to avoid scratching the array surface. 
Protein probe not applied properly Apply the probe solution and LifterSlip™ or equivalent coverslip to the array as described in the manual. To avoid drying of the array surface, make sure the coverslip covers the printed area of the array and adjust the coverslip, if needed. 
Probe or detection reagents contain precipitates Centrifuge the probe or detection reagents to remove precipitates prior to probing the array. 

Protoarray™Microarray—Kinase Substrate Identification (KSI) Application

KSI アプリケーションで Protoarray™ マイクロアレイを使用したところ、タンパク質キナーゼで得られたシグナルは非常に弱いかまたは全くありませんでした。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Kinase of interest is not active or is inactivated by the assay buffer Check the activity of the kinase after purification using a method of choice. Ensure the kinase is active under the conditions used for probing. 
Avoid repeated freezing-thawing of your kinase. 
Low specific activity of the kinase Perform probing with higher kinase concentration, higher kinase specific activity, or increase the incubation time. 
Avoid repeated freezing-thawing of your kinase. 
Incorrect scanning or imaging 

For X-ray film, develop the film and acquire the image using a standard scanner. 

For phosphor screen, acquire the image using a phosphorimager. Follow the manufacturer’s recommendations on using the scanner or phosphor imager to scan the array correctly. Be sure to use a scanner or phosphorimager that provides at least 50 μM resolution and generates 16-bit TIFF image files. 

Incorrect assay conditions Perform incubation of the array at 30 degrees C during the probing procedure. 
Use freshly prepared Kinase Buffer for best results. 
Poor incorporation of radiolabel Use fresh [γ33P]ATP. Be sure to check the array using a Geiger counter to verify that the radioactive signal is obtained after the probing procedure. 
Kinase-ATP mixture not added immediately to the array After preparing the kinase-ATP mixture, immediately add the mixture to the array. Do not store the prepared kinase-ATP mixture on ice for more than 2 minutes prior to use on the array. 
Kinase specific substrates are not present on the array Use another kinase. 
KSI アプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に高いバックグラウンドが得られました。 どのような解決策が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Improper blocking Prepare the KSI Blocking Buffer freshas described on page 38 of the manual.
Improper washing For the best results, perform the recommended washing steps using 0.5% SDS and water as outlined in the protocol. 
Array dried during probing or washing Do not allow the array to dry during probing or washing procedure. 
Ensure the coverslip completely covers the printed area of the array. During the incubation step at 30 degrees C, make sure the 50-mL conical tube is capped to minimize drying. 
During all wash steps, ensure the array is completely covered in buffers. 
Array not dried properly before scanning Dry the array as described before scanning. 
High kinase concentration Decrease the kinase concentration/specific activity or decrease the incubation time. 
KSI アプリケーションで Protoarray™ マイクロアレイを使用していますが、不均一なバックグラウンドが得られます。 何かヒントはありますか？

Here are possible causes and solutions:

Cause

Solution

Uneven blocking or washing During the blocking or washing steps, ensure the array is completely immersed in buffers and use at least 40 mL buffer in the 50-mL conical tube to cover the array completely with buffer. 
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the 0.5% SDS solution fresh as described on page 38 of the manual.
Portions of array have dried Do not allow the array to dry during probing. 
Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps. Take care while inserting the array into the tube to avoid scratching the array surface. 
Radiolabeled ATP or buffer contains precipitates Centrifuge the [γ33P]ATP or buffer to remove precipitates prior to probing the array. 
KSI アプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に解像度の低いスポットが得られました。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Incorrect scanner or phosphor imager used Be sure the scanner or phosphor imager is capable of providing at least 50 μM resolution. 
Improper handling of arrays Be sure to allow the mailers with arrays to equilibrate at 4 degrees C for at least 15 minutes prior to use. 
Improper covering of arrays Properly cover the array with a single layer of clear plastic wrap without any creases. 
KSI アプリケーションで Protoarray™ マイクロアレイを使用したところ、二重反復（2 連）スポットからのシグナルが一体化していることに気づきました。 何が起きたのでしょうか？

It is normal for signals from duplicate spots to merge sometimes. The merging of spots does not affect data analysis. 

Protoarray™ Microarray—Small Molecule Identification (SMI)-Fluorescent Application

SMI 蛍光アプリケーションで Protoarray™ マイクロアレイを使用したところ、低分子プローブで得られたシグナルは非常に弱いかまたは全くありませんでした。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Epitope tag not present Confirm the presence of the tag by appropriate assay. 
Poor biotinylation of small molecule Make sure the small molecule is in a buffer that does not contain any primary amines such as ammonium ions, Tris, glutathione, imidazole, or glycine. 
Make sure the biotinylation reaction was performed correctly using the specified molar ratios and at pH ~8.0. Check that the calculations and serial dilutions are performed correctly. 
Low probe concentration Perform probing with higher probe concentration or increase the incubation time. 
Incorrect probing procedure Follow the recommended protocols for probing on pages 57 and 58 of the manual. Be sure all incubations are performed at 4 degrees C. Prepare the SMI Assay Buffer fresh as described on page 54 of the manual. 
Do not allow the array to dry during the probing procedure. 
Avoid prolonged exposure of detection reagents labeled with a fluorescent dye to light. 
Incorrect scanning or imaging Scan the array at suitable wavelength for the detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source. 
Decrease stringency Decrease the number of washes. Perform probing and washing in the absence or lower concentration of detergent or salts. 
SMI 蛍光アプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に高いバックグラウンドが得られました。 どのような解決策が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Improper blocking Prepare the SMI Assay Buffer freshas described on page 54 of the manual.
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the SMI Assay Buffer freshas described on page 54 of the manual.
Array dried during probing Do not allow the array to dry during probing. 
Array not dried properly before scanning Dry the array as described on page 59 of the manual before scanning. 
High probe concentration Decrease the probe concentration or decrease the incubation time. 
SMI 蛍光アプリケーションで Protoarray™ マイクロアレイを使用していますが、不均一なバックグラウンドが得られます。 何かヒントはありますか？

Here are possible causes and solutions:

Cause

Solution

Uneven blocking or washing During the blocking or washing steps, ensure the array is completely immersed in SMI Assay Buffer and use at least 5 mL buffer in the incubation tray to cover the array completely with buffer. 
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the SMI Assay Buffer fresh as described on page 54 of the manual.
Portions of array have dried Do not allow the array to dry during probing. 
Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps. Take care while inserting the array into the tube to avoid scratching the array surface. 
Small molecule probe not applied properly Apply the probe solution and LifterSlip™ or equivalent coverslip to the array as described in the manual. To avoid drying of the array surface, make sure the coverslip covers the printed area of the array and adjust the coverslip, if needed. 
Probe or detection reagents contain precipitates Centrifuge the probe or detection reagents to remove precipitates prior to probing the array. 

Protoarray™ Microarray—Small Molecule Identification (SMI)-Radioactive Application

SMI 放射活性アプリケーションで Protoarray™ マイクロアレイを使用したところ、低分子プローブで得られたシグナルは非常に弱いかまたは全くありませんでした。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Low specific activity of the small molecule Perform probing with higher small molecule concentration, higher small molecule specific activity, or increase the incubation time. 
Incorrect scanning or imaging 

Follow the manufacturer’s recommendations on using the scanner or phosphor imager to scan the array correctly. For phosphor screen, acquire the image using a phosphor imager. 

Be sure to use a scanner or phosphor imager that provides at least 50 μM resolution and generates 16-bit TIFF image files. 

Incorrect assay conditions Perform incubation of the array at 4 degrees C during the probing procedure. Use freshly prepared Tritium SMI Assay Buffer for best results. 
Small molecule specific substrates are not present on the array Use another small molecule. 
SMI 放射活性アプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に解像度の低いスポットが得られました。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Incorrect phosphor imager used Be sure the phosphor imager is capable of providing at least 50 μM resolution. 
Improper handling of arrays Be sure to allow the mailers with arrays to equilibrate at 4 degrees C for at least 15 minutes prior to use. 
SMI 放射活性アプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に高いバックグラウンドが得られました。 どのような解決策が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Improper blocking Prepare the Tritium SMI Assay Buffer freshas described on page 68 of the manual.
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the SMI Assay Buffer freshas described on page 68 of the manual.
Array dried during probing or washingDo not allow the array to dry during probing or washing. 
Ensure the coverslip completely covers the printed area of the array. During the incubation step at 4 degrees C, make sure the 50-mL conical tube is capped to minimize drying. 
During all wash steps, ensure the array is completely covered in buffers. 
Array not dried properly before scanning Dry the array as described on page 72 of the manual before scanning.
High probe concentration Decrease the probe concentration/specific activity or decrease the incubation time. 
SMI 放射活性アプリケーションで Protoarray™ マイクロアレイを使用していますが、不均一なバックグラウンドが得られます。 何かヒントはありますか？

Here are possible causes and solutions:

Cause

Solution

Uneven blocking or washing During the blocking or washing steps, ensure the array is completely immersed in buffers and use at least 40 mL buffer in the 50-mL conical tube to cover the array completely with buffer. 
Improper washing To obtain the best results, perform the recommended washing steps. Prepare Tritium SMI Assay Buffer fresh as described on page 68 of the manual.
Portions of array have dried Do not allow the array to dry during probing. 
Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps. Take care while inserting the array into the tube to avoid scratching the array surface. 
Reagents or buffer contains precipitates Centrifuge the reagents or buffer to remove precipitates prior to probing the array. 
SMI 放射活性アプリケーションで Protoarray™ マイクロアレイを使用したところ、二重反復（2 連）スポットからのシグナルが一体化していることに気づきました。 何が起きたのでしょうか？

It is normal for signals from duplicate spots to merge sometimes. The merging of spots does not affect data analysis.

Protoarray™ Microarray—Ubiquitin Ligase Profiling Application

ユビキチンリガーゼプロファイリングアプリケーションで Protoarray™ マイクロアレイを使用したところ、タンパク質プローブで得られたシグナルは非常に弱いかまたは全くありませんでした。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Low enzyme concentrations Perform probing with higher enzyme concentrations or increase the incubation time. 

Incorrect probing procedure

Follow the recommended protocols for probing on page 84 of the manual. Be sure all incubations are performed at the appropriate temperatures. Prepare the Assay Buffer fresh as described on page 82 of the manual.
Do not allow the array to dry during the probing procedure.
Avoid prolonged exposure of detection reagents labeled with a fluorescent dye to light.
Incorrect scanning or imaging Scan the array at suitable wavelength for the detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source. 
Decrease stringency Decrease the number of washes. Perform probing and washing in the absence or lower concentration of detergent or salts. 
ユビキチンリガーゼプロファイリングアプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に高いバックグラウンドが得られました。 どのような解決策が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Improper blocking Prepare the Blocking Buffer freshas described on page 82 of the manual.
Improper washing To obtain the best results, perform the recommended washing steps. Prepare 0.5% SDS solution fresh as described on page 81 of the manual.
Array dried during probing Do not allow the array to dry during probing.
Array not dried properly before scanning Dry the array as described on page 85 of the manual before scanning. 
High enzyme concentration Decrease the enzyme concentration or decrease the incubation time. 
ユビキチンリガーゼプロファイリングアプリケーションで Protoarray™ マイクロアレイを使用していますが、不均一なバックグラウンドが得られます。 何かヒントはありますか？

Here are possible causes and solutions:

Cause

Solution

Uneven blocking or washing During the blocking or washing steps, ensure the array is completely immersed in Blocking Buffer or Assay Buffer, and use at least 5 mL buffer in the incubation tray to cover the array completely with buffer. 
Improper washing To obtain the best results, perform the recommended washing steps. Prepare
0.5% SDS solution fresh as described on page 81 of the manual.
Portions of array have dried Do not allow the array to dry during probing. 
Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps. Take care while inserting the array into the tube to avoid scratching the array surface. 
Protein probe not applied properly Apply the probe solution and LifterSlip™ or equivalent coverslip to the array as described in the manual. To avoid drying of the array surface, make sure the coverslip covers the printed area of the array and adjust the coverslip, if needed. 
Probe or detection reagents contain precipitates Centrifuge the probe or detection reagents to remove precipitates prior to probing the array. 

Protoarray™ Microarray—Immune Response Biomarker Profiling (IRBP) Application

IRBP アプリケーションで Protoarray™ マイクロアレイを使用したところ、血清サンプルで得られたシグナルは非常に弱いかまたは全くありませんでした。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Low serum concentration Perform probing with higher serum concentration or increase the incubation time. 
Incorrect probing procedure Follow the recommended protocol for probing. Be sure all incubations are performed at 4 degrees C. Prepare the Blocking Buffer and Washing Buffer fresh as described on page 94 of the manual.

Avoid prolonged exposure of detection reagents labeled with fluorescent dye to light. 

Incorrect scanning or imaging Scan the array at suitable wavelength for the detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source. 
Decrease stringency Decrease the number of washes. Perform probing and washing in the absence or lower concentration of detergent or salts. 
IRBP アプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に高いバックグラウンドが得られました。 どのような解決策が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Improper blocking Prepare the Blocking Buffer fresh as described on page 94 of the manual.
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the Washing Buffer fresh as described on page 94 of the manual.
Array dried during probing Do not allow the array to dry during probing. 
Array not dried properly before scanning Dry the array before scanning. 
High serum concentration Decrease the serum concentration or decrease the incubation time. 
Antibody cross-reactivity Probe a protein array using only the secondary antibody without the serum sample to detect cross-reactivity with the antibody only. 
IRBP アプリケーションで Protoarray™ マイクロアレイを使用していますが、不均一なバックグラウンドが得られます。 何かヒントはありますか？

Cause

Solution

Uneven blocking or washing During the blocking or washing steps, ensure the array is completely immersed in blocking solution or Washing Buffer and use 5 mL buffer in each chamber of the incubation tray to cover the array completely with buffer. 
Improper washingTo obtain the best results, perform the recommended washing steps. Prepare the Washing Buffer fresh as described on page 94 of the manual.
Portions of array have dried Do not allow the array to dry during probing. 
Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps. Take care while inserting or removing the array from the incubation tray to avoid scratching the array surface. 
Serum sample or detection reagents contain precipitates Centrifuge the serum sample or detection reagents to remove precipitates prior to probing the array. 

Protoarray™ Microarray—Antibody Specificity Profiling Application

抗体特異的プロファイリングアプリケーションで Protoarray™ マイクロアレイを使用したところ、抗体で得られたシグナルは非常に弱いかまたは全くありませんでした。 原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Low antibody concentration Perform probing with higher antibody concentration or increase the incubation time. 
Incorrect probing procedure Follow the recommended protocol for probing. Be sure all incubations are performed at 4 degrees C. Prepare the Blocking Buffer and Washing Buffer fresh as described on page 106 of the manual.

Avoid prolonged exposure of detection reagents labeled with fluorescent dye to light. 

Incorrect scanning or imaging Scan the array at suitable wavelength for the detection system used and place the array in the slide holder such that the proteins on the array are facing the laser source. 
Decrease stringency Decrease the number of washes. Perform probing and washing in the absence or lower concentration of detergent or salts. 
抗体特異的プロファイリングアプリケーションで Protoarray™ マイクロアレイを使用したところ、非常に高いバックグラウンドが得られました。 どのような解決策が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Improper blocking Prepare the Blocking Buffer fresh as described on page 106 of the manual.
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the Washing Buffer fresh as described on page 106 of the manual.
Array dried during probing Do not allow the array to dry during probing. 
Array not dried properly before scanning Dry the array before scanning. 
High antibody concentration Decrease the antibody concentration or decrease the incubation time. 
Antibody cross-reactivity Probe a protein array using only the secondary antibody without the primary antibody sample to detect cross-reactivity with the secondary antibody only. 
抗体特異的プロファイリングアプリケーションで Protoarray™ マイクロアレイを使用していますが、不均一なバックグラウンドが得られます。 何かヒントはありますか？

Here are possible causes and solutions:

Cause

Solution

Uneven blocking or washing During the blocking or washing steps, ensure the array is completely immersed in blocking solution or Washing Buffer and use 5 mL buffer in each chamber of the incubation tray to cover the array completely with buffer. 
Improper washing To obtain the best results, perform the recommended washing steps. Prepare the Washing Buffer fresh as described on page 106 of the manual. 
Portions of array have dried Do not allow the array to dry during probing. 
Improper array handling Always wear gloves and avoid touching the surface of the array with gloved hands or forceps. Take care while inserting or removing the array from the incubation tray to avoid scratching the array surface. 
Antibody sample or detection reagents contain precipitates Centrifuge the antibody sample or detection reagents to remove precipitates prior to probing the array. 

For Research Use Only. Not for use in diagnostic procedures.