Q: What types of studies can I conduct using the HuProt array?
A: The HuProt array and subsets of the HuProt have been used successfully in a range of applications, which include:
Ref: Hu C et al.(2015) Pubmed
Antibody cross-reactivity testing
Ref: Venkataraman A et al.(2018) Pubmed
Ref: Deng RP et al.(2014) Pubmed
Ref: Hu S et al.(2009) Pubmed
Ref: Barry G et al.(2014) Pubmed
Ref: Cox et al.(2015) Pubmed
HuProt Storage, Stability, and Documentation:
Q: What is the expected shelf life of the HuProt arrays? How should they be stored, and how are they shipped?
A: If the HuProt arrays are properly stored at -80˚C, the arrays have a shelf life of at least 6 months for most applications. For serum profiling, the arrays are good for at least one year after the date of manufacture.
Q: Are the arrays labile, and how is that measured?
A: The proteins printed on the arrays are very stable if the arrays are stored at -80˚C. We have performed studies using arrays that are 24 months old and still observed significant binding signals.
Q: Can I obtain SOPs and a MSDS for HuProt arrays?
A: User manual and a MSDS are available for HuProt arrays in the above archive.
Q: Are HuProt arrays CGMP (Current Good Manufacturing Practice) compliant?
A: HuProt arrays are used primarily as for in vitroresearch and are not CGMP compliant.
Q: What is the maximum order volume per month? How long is the shipping/delivery time after receiving the order?
A: Please contact us – we will be happy to discuss your inventory needs with you.
Q: How are the HuProt arrays shipped?
A: HuProt arrays are placed in plastic slide mailers, which are in turn securely boxed and shipped on dry ice.
Q: I am an overseas client and need a copy of the Commercial Invoice in order to import the arrays. Can you send us a copy?
A: If you need a copy of the commercial invoice for customs, please let us know and we will send you a copy in advance.
Q: Where can I find the full range of proteins on the HuProt array?
A: For the full list of proteins by name and category please visit the resources section above.
Q: How are the full-length proteins attached to the glass surface of the arrays?
A: Standard HuProt arrays are printed on nitrocelluose coated PATH™ slides. Other surfaces may be available, depending on your needs. Please contact us to discuss.
Q: Is new content added to the HuProt array on a regular basis (e.g. every 6 months)
A: We periodically add new proteins to the array when new full-length cDNA clones are made available.
Q: Are all proteins printed in equal amounts on the HuProt arrays? Are proteins that are normally abundant in the cell normalized to levels similar to the less abundant?
A: At present, the amount of protein on the array at present is not normalized, and varies depending on the protein level expressed in yeast prior to purification. In some rare cases, the protein levels may be low if the expressed protein is toxic to the yeast cells.
Q: Are membrane proteins represented on the array? If so, is there some detergent present?
A: The HuProt array contains thousands of membrane proteins. The protein printed on the arrays is eluted using a buffer that contains 0.03% TritonX-100.
Q: The array is spotted with full-length protein containing GST tags. Does that affect the conformation of proteins with transmembrane domains?
A: We extensively evaluated the folding of many non-membrane proteins on the HuProt array. However, we did not perform detailed analysis on the folding of membrane proteins. The full protein content on the HuProt arrays is found at the top of this page. The full protein content on the HuProt arrays can be found at the top of this page.
Q: Have the arrays been tested for pH tolerance? For example, I am interested in studying post-translational modifications, which work best at a higher pH > 8.0.
A: All our proteins were eluted in buffer of pH 8.0–pH8.5 and should work well for studies of post-translational modifications.
Q: Have different buffers been tested on the HuProt array?
A: Our arrays have been used successfully in a range of different buffers to study serum profiling, protein binding, DNA binding, RNA binding, kinase assay, acetylation, SUMOylation/ubiquitinylation, etc. Please contact us to discuss specifics regarding your assay and buffer.
Q: I am new to protein array work. Are slides with just the control spots available for pre-testing and practice? It would be nice to also practice scanning before using actual HuProt arrays
A: While we currently don’t offer test arrays that contain just controls spots, these are under development. You may practice the steps in your assay with a standard glass microscope prior to using the HuProt arrays. Regarding scanning, the GenePix Pro software has an “auto-PMT” function.
Q: Can the HuProt Arrays be stripped and re-used? I don’t require active proteins for my study.
A: At CDI we do not strip or reuse HuProt arrays. If you wish to test your sample against inactive proteins, however, you are welcome to try stripping the array.
Q: I would like CDI to add some proteins that are not printed on the HuProt array to my order. How much protein should I send to CDI Labs?
A: Please send at least 10 µl of protein–the protein must have a concentration of at least 300ng/µl. We will dilute the protein to 150 ng/µl in printing buffer and then print it on the HuProt arrays ordered. An additional fee will be charged for this service. Please allow additional time for these customized arrays to be shipped to you.
HuProt Production and Quality Control:
Q: What kind of QC is done on the HuProt array by CDI? Is a QC report for production available?
A: Each batch of HuProt arrays must pass rigorous QC requirements, and comes with a certificate of analysis.
Q: What QC methods are used to determine good/passing protein printing on the arrays?
A: To meet CDI Labs QC standards, >95% of the spots must generate GST signals with a foreground/background ratio >1.5.
Q: Can you tell me more about the variability within a specific lot of HuProt arrays?
A: Our QC protocol includes sampling at specific points across each print run, to ensure that quality is consistent throughout each printed batch.
HuProt Array Controls:
Q: What control spots are printed on HuProt slides?
A: The HuProt array contains 16 landmarks, including fluorophores, as follows:
- H1 – Histone H1
- H2 (A+B) – Histone H2A and H2B mixture
- H3 – Histone H3
- H4 – Histone H4 (all the histones are non-specific binding proteins, used as positive controls for a variety of assays.)
- IgG488/594 – Alexa Fluor 488/594 labeled IgG, positive control and landmarks for fluorescent detection in 488/594 channels.
- IgG555/647 – Alexa Fluor 555/647 labeled IgG, positive control and landmarks for fluorescent detection in 532/635 channels.
- GST10n – glutathione S-transferase at 10 ng/μl
- GST50n – GST at 50 ng/μl
- GST100n – GST at 100 ng/μl
- GST200n – GST at 200 ng/μl
- Mouse-anti-biotin – positive control for biotinylated samples and anti-mouse IgG detection
- Rabbit-anti-biotin – positive control for biotinylated samples and anti-rabbit IgG detection
- Biotin-BSA – biotinylated BSA, positive control for streptavidin detection
- BSA – Bovine serum albumin, negative control
- Buffer – printing buffer only, negative control
- Mouse IgM – positive control for anti-mouse-IgM detection
Q: Are there reference spots (landmarks), such as GFP, that are spotted regularly on the arrays?
A: There is one row of control spots at the bottom of each block, including Alexa Fluor 555/647 fluorescence as landmarks, but no GFP.
HuProt Data Analysis:
Q: What scanner should I use to analyse the array data?
A: The HuProt array is entirely compatible with a range of array scanners. At CDI Labs we employ a GenePix scanner and software for in-house testing services. Yes, we do supply a .gal file for each lot of our arrays, for data generation.
Q: Is there a general analysis methodology, with best practices, for analyzing HuProt microarray data?
A: We analyze scanned array images using GenePix Pro software to get a raw data file (.gpr files). The Z-score of each spot on the array is calculated according to this algorithm: α= Foreground (sample channel), e.g. F635 z = [α – α(avg)] / α(std) α(avg)] and α(std) are the average and standard deviation of α values of all spots on the array. A protein is considered as positive (positive hit) if the average Z-score of its duplicate spots is > 3.
Q: What are the best normalization practices?
A: The algorithm above uses the average signal intensity of all spots on the array for normalization.
Q: Aside from the GenePix Pro software, are there other useful software analysis programs?
A: CDI Labs finds that GenePix Pro is the best software for analysis. We only use the Z-score of the average normalized signal intensity to rank hits.
Q: In the Z-score algorithm, how is the average alpha value calculated? Is this the average of duplicates for a protein feature?
A: The average alpha value is of all the spots on a given array.
Q: There is an output function in the .gpr file that lists the signal-to-noise (SNR) for both channels. This is similar to the Z-score you mentioned. Is this what you are calling the Z-score, and why is the “positive hit” threshold set at >3?
A: No. We use the algorithm described above to calculate the average Z-score but not the SNR in the .gpr file.
Q: What can we expect for detection sensitivity? At what Kd can we expect resilient detection, and at what Kd range does detection drop off?
A: For protein-DNA interactions the affinity is ~ 500 nM. For protein-protein interactions the affinity can be as low as 1 μM. We have also tested many antibodies with affinities ranging from 500 nM to 0.1 nM. We have not systematically defined the Kd range at which detection drops off.
Q: Is there an internal standard for protein-protein interactions to account for potential false negatives?
A: The HuProt array does not include internal standards specific to PPI, but histones are used as positive controls for most applications, including protein-protein interactions. HuProt array is a discovery tool and we have always find dozens, if not hundreds, of positive hits on HuProt – false negatives are not considered a significant issue.
Q: For protein-protein interaction studies, is there an internal standard to account for potential false negatives?
A: The HuProt array uses histones as positive controls for most applications, and does not include internal standards specific to protein-protein interactions. CDI has found that false negatives are not a significant issue in HuProt studies.
GST Staining & Gridding:
Q: I would like to co-stain the array with labeled anti-GST antibody when we add our antibody of interest. What anti-GST concentration do you recommend if I use 1.0 μg/mL of our antibody of interest?
A: We recommend adding only 0.1 μg/ml of anti-GST to minimize the competition between anti-GST and the mAb (sample) that is being tested. While this will result in weak GST signals, these are strong enough for grid alignment. If you wish to see strong GST signals, you may use 0.5-1.0 μg/ml of anti-GST antibody; however, we have observed a few cases where the secondary antibody cross-reacted with the anti-GST antibody, which resulted in similar signal patterns in the sample channel and the GST channel. If you are familiar with the grid alignment, we encourage you to run the sample mAb only (without anti-GST), or re-probe the array with anti-GST after scanning your antibody signals.
Q: What are the units for the GST controls (i.e. GST10n)? What is “n?”
A: The “n” is ng/μl.
Q: Can the GST control information be used to analyze microarray data in regard to the other protein spots (both the anti-GST signal channel and signals from our protein of interest)?
A: No. The anti-GST information is used to facilitate grid alignment prior to data analysis, but is not used for QC or for normalization of the array data. Our tests using this approach found that many spots with very low GST signals are amplified, so we do not recommend GST normalization for array data analysis (please refer to the list of landmarks above).
Q: If we don’t observe anti-GST signals at all positions on the microarray, what does this mean? Was there not enough anti-GST present? Or was the protein not printed on that spot?
A: The anti-GST antibodies are added only to facilitate grid alignment, therefore only a very low concentration of anti-GST antibody is added (1:10,000 dilution). This minimizes the competition between the anti-GST antibodies and the actual sample being studied. As a result, the GST signals may appear weak or even invisible on some spots. Please note that not all controls contain a GST tag (please refer to the list of landmarks listed).
Q: Your protocol specifies re-probing with anti-GST to establish the reference grid. Should I strip off the first label before adding the second probe? May I probe the array with anti-GST and my primary antibody at the same time, followed by both secondary antibodies at the same time? This would let me simultaneously scan both antibodies and save processing time.
A: No, there is no need to strip away the first antibody before re-probing with anti-GST. As long as there are no concerns that the anti-GST could interfere with the primary antibody, you may add both probes simultaneously to the array, and then add the different secondary antibodies simultaneously to detect hits.
Q: Where can I obtain an image of a good quality anti-GST array stain, to compare to our results? What were the staining conditions?
A: GST images are available for each batch printed, and can be downloaded from the CDI website Resources page. (http://cdi-lab.com/resources.shtml): Typically, we use a 1:2,000 dilution of rabbit anti-GST as the primary antibody, and an Alexa555-anti-Rabbit as the secondary antibody. The blocking buffer is PBS-T with 5% BSA, and the staining protocol used is as stipulated in the users’ manual.
Antibody Specificity and Cross-Reactivity Testing
Q: What concentration is needed for Monoclonal Antibody (mAb) Specificity Testing on the HuProt array?
A: To test mAb specifity, a concentration of 0.1 – 1.0 μg/ml (depending on the mAb affinity) is recommended.
Q: What concentration of mAb do I use to see if it cross-reacts with other proteins on the array other than the antigen?
A: To test the cross-reactivity of your mAb against other proteins on the array, increase the mAb concentration in your assay to 10 μg/ml.
Q: How many monoclonal antibodies can I test on each array for cross-reactivity? Can the arrays be used more than once? How about competition studies?
A: We recommend that each array is used only once for cross-reactivity testing, as the printed proteins will denature during the drying process done prior to scanning. For competition studies, you may add 2 mAbs at the same time to one array (0.1 – 1.0 μg/ml of each mAb).
Q: For antibody cross-reactivity testing, when should I add anti-GST antibodies for grid alignment, and which scanner wavelength should I use for anti-GST staining?
A: For analyses using one mAb sample per array, to view the anti-GST staining use a wavelength different from that used to stain your mAb. There are two ways to do this:
- Add the anti-GST antibodies and mAb sample to the array at the same time (not recommended).
- To prevent the anti-GST antibody from competing with your sample mAb or cross-reacting with the secondary antibody, first perform the assay using your sample mAb and then scan the array (most protein spots will not be visible). Next, add the anti-GST antibodies to the array, and then apply the grid pattern to the mAb image. The GST signals will be weak as low concentrations of anti-GST are used. For more information, refer to the user manual above.
Q: How much serum should I send to CDI?
A: Please send 50 μl of frozen serum per sample for analysis, preferably on dry ice. As repeating freezing and thawing may affect assay results, please divide the samples into two aliquots, in case test dilutions or a repeat of the assay are needed.
Q: What dilution of serum do you usually use for serum profiling?
A: We typically use a 1:500 or 1:1000 dilution to ensure that the background is low.