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

I purified my protein of interest, which I would like to use as a probe in your Protoarray™ Microarray PPI application, and got no signal after western detection using an antibody against my protein of interest. What could have happened?

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. 

I am trying to biotinylate my protein to use as a probe for the PPI application but I am getting very poor or even no biotinylation. What is the issue?

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. 

I am trying to biotinylate my protein to use as a probe for the PPI application but I am observing additional biotinylation bands. What is happening?

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. 

I probed the Protoarray™ Microarray with my protein probe and got a very weak to no signal. Can you help me troubleshoot?

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. 
I probed the Protoarray™ Microarray with my protein probe and saw very high background. Can you help?

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. 
I probed the Protoarray™ Microarray with my protein probe and got very uneven background. Can you offer some tips?

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

I used your Protoarray™ Microarray in the KSI application and got a very weak to no signal with my protein kinase. What could have gone wrong?

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. 
I used your Protoarray™ Microarray in the KSI application and got a very high background. Can you help me troubleshoot?

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. 
I am using your Protoarray™ Microarray in the KSI application and am getting an uneven background. Can you offer some tips?

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. 
I used your Protoarray™ Microarray in the KSI application and got very poor resolution of spots. What could have gone wrong?

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. 
I used your Protoarray™ Microarray in the KSI application and noticed that signals from duplicate spots were merged. What could have happened?

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

I used your Protoarray™ Microarray in the SMI-fluorescent application and got a very weak to no signal with my small molecule probe. What could have gone wrong?

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. 
I used your Protoarray™ Microarray in the SMI-fluorescent application and got a very high background. Can you help me troubleshoot?

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. 
I am using your Protoarray™ Microarray in the SMI-fluorescent application and am getting an uneven background. Can you offer some tips?

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

I used your Protoarray™ Microarray in the SMI-radioactive application and got a very weak to no signal with my small molecule probe. What could have gone wrong?

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. 
I used your Protoarray™ Microarray in the SMI-radioactive application and got very poor resolution of spots. What could have gone wrong?

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. 
I used your Protoarray™ Microarray in the SMI-radioactive application and got a very high background. Can you help me troubleshoot?

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. 
I am using your Protoarray™ Microarray in the SMI-radioactive application and am getting an uneven background. Can you offer some tips?

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. 
I used your Protoarray™ Microarray in the SMI-radioactive application and noticed that signals from duplicate spots were merged. What could have happened?

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

I used your Protoarray™ Microarray in the ubiquitin ligase profiling application and got a very weak to no signal with my protein probe. What could have gone wrong?

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. 
I used your Protoarray™ Microarray in the ubiquitin ligase profiling application and got a very high background. Can you help me troubleshoot?

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. 
I am using your Protoarray™ Microarray in the ubiquitin ligase profiling application and am getting an uneven background. Can you offer some tips?

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

I used your Protoarray™ Microarray in the IRBP application and got a very weak to no signal with my serum sample. What could have gone wrong?

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. 
I used your Protoarray™ Microarray in the IRBP application and got a very high background. Can you help me troubleshoot?

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. 
I am using your Protoarray™ Microarray in the IRBP application and am getting an uneven background. Can you offer some tips?

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

I used your Protoarray™ Microarray in the antibody specificity profiling application and got a very weak to no signal with my antibody. What could have gone wrong?

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. 
I used your Protoarray™ Microarray for antibody specificity profiling application and got a very high background. Can you help me troubleshoot?

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. 
I am using your Protoarray™ Microarray in the antibody specificity profiling application and am getting an uneven background. Can you offer some tips?

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. 

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