1.

HuProt Applications

HuProt 应用

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:

Serum profiling (Hu C et al. 2015)

Antibody cross-reactivity testing (Venkataraman A et al. 2018)

Protein-protein interaction (Deng RP et al. 2014)

DNA-protein interaction (Hu S et al. 2009)

RNA-protein interaction (Barry G et al. 2014)

Post-translational modification (Cox et al. 2015)

问：HuProt芯片可应用于哪些类型的研究？

答：HuProt大芯片和HuProt的小芯片已成功用于一系列应用，其中包括：

血清分析（Hu C et al. 2015）

抗体交叉反应测试（Venkataraman A et al. 2018）

蛋白质-蛋白质相互作用（Deng RP et al. 2014）

DNA-蛋白质相互作用（Hu S et al. 2009）

RNA-蛋白质相互作用（Barry G et al. 2014）

翻译后修饰（Cox et al. 2015）

2.

HuProt Storage, Stability, and Documentation

HuProt 存储、稳定性和文档

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.

问：HuProt芯片的保质期是多久？如何存储，如何运输？

答：如将HuProt芯片在-80˚C下储存，对于大多数应用，芯片的保质期至少为6个月。对于血清分析，芯片在生产后至少可以使用一年。

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.

问：芯片是否不稳定，如何测定？

答：如果芯片储存在-80˚C，点制在芯片上的蛋白质非常稳定。我们用24个月的芯片进行了检测，仍然可观察到清晰的结合信号。

Q: Are HuProt arrays CGMP (Current Good Manufacturing Practice) compliant?

A: HuProt arrays are used primarily as for in vitro research and are not CGMP compliant.

问：HuProt芯片是否符合CGMP（当前的优良制造规程）？

答：HuProt芯片主要用于体外研究，不符合CGMP要求。

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.

问：每月最大订单量是多少？收到订单后的运输/交货时间需多长时间？

答：请联系我们—我们很乐意讨论您的库存需求。

3.

HuProt Shipping

HuProt 运输

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.

问：HuProt芯片是如何运输的？

答：HuProt芯片放置在塑料载玻片盒中，固定放置运输箱里并放上干冰运输。

4.

HuProt Content

HuProt 内容

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.

问：如何获得HuProt芯片上的全部蛋白质清单？

答：有完整的按名称和类别排列的蛋白质的清单，请访问资源部分。

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.

问：全长蛋白质如何附着在芯片的玻璃表面？

答：标准HuProt芯片点制在硝酸纤维素涂层的PATH™载玻片上。如您需要其他表面，请联系我们。

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.

问：是否定期向HuProt芯片添加新的蛋白质（例如每6个月）

答：如果有新的全长cDNA克隆可用，我们会定期将新蛋白质添加到芯片中。

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.

问：在HuProt芯片上点制的蛋白质是否都是等量的？在细胞中的高丰度蛋白质是否调整到与低丰度的蛋白质相似的水平？

答：目前芯片上的蛋白量没有标准化，根据纯化前酵母表达的蛋白水平而有所不同。在极少数情况下，如果表达的蛋白质对酵母细胞有毒，则蛋白质的量可能会很低。

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.

问：芯片上是否有膜蛋白？如果有，是否含有洗涤剂？

答：HuProt芯片包含数千种膜蛋白，点制在芯片上的蛋白质是用0.03% TritonX-100缓冲液洗脱。

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.

问：芯片上的蛋白质是带有GST标签的全长蛋白质，这会影响具有跨膜结构域的蛋白质的构象吗？

答：我们广泛地评估了HuProt芯片上许多非膜蛋白的折叠，然而，我们没有对膜蛋白的折叠进行详细分析。

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.

问：芯片是否经过了pH耐受性测试？例如，我对翻译后修饰研究感兴趣，这种修饰在pH >8.0时效果较好。

答：所有的蛋白质都在pH 8.0–8.5的缓冲液中洗脱，应该可以适合于翻译后修饰的研究。

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.

问：是否在 HuProt 芯片上测试过不同的缓冲液？

答：芯片已成功用于一系列不同的缓冲液中，用以研究血清分析、蛋白质结合、DNA结合、RNA结合、激酶测定、乙酰化、SUMO化/泛素化等。请联系我们具体讨论不同检测和缓冲液。

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.

问：我是蛋白质芯片工作的新手，有没有只有对照点的载玻片可用于预实验和练习？在使用实际的HuProt芯片之前能练习扫描会很有帮助。

答：我们目前不提供仅包含对照点的测试芯片，这些正在开发中。在使用HuProt芯片之前，您可以用标准的显微镜载玻片做练习。关于扫描，GenePix Pro软件具有“自动 PMT”功能。

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.

问：HuProt芯片可以漂洗并再利用吗？我的研究不需要活性蛋白质。

答：我们一般不会漂洗或再利用HuProt芯片。但如果您希望针对非活性蛋白质测试您的样品，欢迎您进行尝试。

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.

问：我希望CDI在HuProt芯片上添加我的蛋白质，应该向CDI实验室提供多少蛋白质？

答：请提供至少10 μL的蛋白质—该蛋白质的浓度至少为300 ng/μL。我们将在点样缓冲液中将蛋白质稀释至150 ng/μL，然后将其点制在定制的HuProt芯片上。此项服务将收取额外费用，请留出更多时间将这些定制芯片寄给您。

Q: Approximately how much of each protein is spotted on the HuProt microarray?

A: Other than controls, the HuProt library is expressed, purified, and spotted ‘as-is’ – meaning we do not know the specific concentration of most proteins printed. Parent protein aliquots are known to range from 0.001-2.5 mg/mL in concentration, with an average of 0.15 mg/mL. Each protein aliquot is printed in duplicate with 100 picoliters (pL) volume per protein spot.

问：点制在HuProt芯片上的蛋白质每种大约有多少？

答：除了对照，HuProt的蛋白质按“原样”表达、纯化和点制——即我们不知道点制蛋白质的具体浓度。已知蛋白质母液的浓度范围为0.001-2.5 mg/mL，平均为 0.15 mg/mL。每个蛋白质点被重复点制在芯片上，每个蛋白质点的体积为100皮升（pL）。

5.

HuProt Production and Quality Control

HuProt 的生产和质量控制

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.

问：HuProt芯片点制中有哪些质控？是否提供生产质量控制报告？

答：每批HuProt芯片都要通过严格的QC检查，并附有分析证书。

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.

问：用哪些QC方法来确定芯片上的蛋白质属于良好/通过？

答：CDI Labs的QC标准是>95%的样品点必须生成前景/背景比率>1.5的GST信号。

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.

问：能告诉我更多关于特定批次HuProt芯片的可靠性吗？

答：我们的QC流程包括在每批点制芯片中取样检查，以确保每个打印批次的质量始终如一。

6.

HuProt Array Controls

HuProt芯片中的对照

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

问：HuProt芯片上有哪些对照点？

答：HuProt芯片包含16个标志点，包括各种荧光点，见下：

H1 – 组蛋白H1

H2 (A+B) – 组蛋白H2A和H2B的混合物

H3 – 组蛋白H3

H4 – 组蛋白H4（所有组蛋白都是非特异性结合蛋白，用作各种检测的阳性对照。）

IgG 488/594 – Alexa Fluor 488/594 标记的IgG、阳性对照和标志物，用于在 488/594 通道中进行荧光检测。

IgG 555/647 – Alexa Fluor 555/647标记的IgG、阳性对照和标志物，用于532/635通道中的荧光检测。

GST10n – 10 ng/μL的谷胱甘肽S-转移酶（GST蛋白质）

GST50n – 50 ng/μL的GST

GST100n – 100 ng/μL的GST

GST200n – 200 ng/μL的GST

鼠抗生物素 – 生物素化样品和抗小鼠IgG检测的阳性对照

兔抗生物素 – 生物素化样品和抗兔IgG检测的阳性对照

生物素-BSA – 生物素化的BSA，链霉亲和素检测的阳性对照

BSA – 牛血清白蛋白，阴性对照

缓冲液 – 只有点样缓冲液，阴性对照

小鼠IgM – 抗小鼠IgM检测的阳性对照

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.

问：芯片上是否有规则排布的参考点（标志点），如GFP？

答：每个block的底部有一排对照点，包括Alexa Fluor 555/647荧光作为标志点，但没有GFP。

7.

HuProt Data Analysis

HuProt 数据分析

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.

问：应该使用什么扫描仪来分析芯片数据？

答：HuProt芯片与一系列芯片扫描仪完全兼容。在CDI实验室，我们使用 GenePix扫描仪和软件进行内部测试服务。我们为每批芯片都会提供一个.gal文件，用于数据分析。

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.

问：是否有用于分析HuProt芯片数据的通用分析方法和最佳实践？

答：我们用GenePix Pro软件分析扫描的芯片图像以获取原始数据文件（.gpr文件）。芯片上每个点的Z分数根据以下算法计算：α = 前景（样本通道），例如F635 z = [α – α(avg)] / α(std)，α(avg)和α(std)是芯片上所有点的α值的平均值和标准偏差。如果蛋白质重复点的平均Z值>3，则该蛋白质被视为阳性（阳性点）。

Q: What are the best normalization practices?

A: The algorithm above uses the average signal intensity of all spots on the array for normalization.

问：最佳的归一化方法是哪种？

答：上述算法是用芯片上所有点的平均信号强度进行归一化。

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.

问：除了GenePix Pro软件，还有其他可用的分析程序吗？

答：CDI Labs认为GenePix Pro是最好的分析软件，我们只使用平均归一化信号强度的Z分数来对阳性点进行排序。

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.

问：在Z-score算法中，平均α值是如何计算的？这是重复蛋白质点的平均值吗？

答：平均α值是给定芯片上所有点的平均值。

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.

问：.gpr文件中有一个输出函数，它列出了两个通道的信噪比（SNR）。这类似于您提到的Z分数，这就是您所说的Z分数吗？为什么“阳性点”阈值设置为>3？

答：不是。我们使用上述算法计算平均Z分数，但不计算.gpr文件中的SNR。

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.

问：我们对检测灵敏度有何期望？可以期望在多大的Kd下进行可重复的检测，以及在多大的Kd范围内检测率会下降？

答：对于蛋白质-DNA相互作用，亲和力约为500 nM，对于蛋白质-蛋白质相互作用，亲和力可低至1 μM。我们还测试了许多亲和力从500 nM到0.1 nM的抗体。我们还没有系统地定义检测率下降的Kd范围。

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.

问：是否有蛋白质-蛋白质相互作用的内标来解释潜在的假阴性？

答：HuProt芯片不包含特定PPI的内标，但组蛋白可用作大多数应用的阳性对照，包括蛋白质-蛋白质相互作用。HuProt芯片是一种发现的技术平台，我们总是在HuProt上发现数十个（甚至数百个）阳性命中-假阴性不被认为是一个重大问题。

8.

GST Staining & Gridding

GST 染色和网格化

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.

问：在使用我们感兴趣的抗体时，还想用标记的抗GST抗体对芯片进行共染色。如果我感兴趣的抗体浓度是1.0 μg/mL，推荐抗GST浓度是多少？

答：我们建议仅加入0.1 μg/mL的抗GST抗体，以减少抗GST与正在测试的 mAb（样品）之间的竞争。虽然这会导致GST信号较弱，但这些信号对于网格对齐来说已经足够强了。如果您希望看到强烈的GST信号，可以用0.5-1.0 μg/mL的抗GST抗体；然而，我们观察到了一些二抗与抗GST抗体发生交叉反应的情况，这导致样品通道和GST通道中的信号模式相似。如果您熟悉网格对齐，我们鼓励您仅使用样品mAb（不使用抗GST），或在扫描抗体信号后再用抗GST抗体探测芯片。

Q: What are the units for the GST controls (i.e. GST10n)? What is “n” ?

A: The “n” is ng/μl.

问：GST对照（即GST10n）的单位是什么？什么是“n”？

答：“n”是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).

问：GST对照信息能否用于分析与其他蛋白质点有关的芯片数据（抗GST信号通道和我们感兴趣的蛋白质的信号）？

答：不可以。抗GST信息用于在数据分析之前保证网格对齐，但不用于QC或芯片数据的归一化。我们对此进行的测试发现，许多低强度的GST信号点被放大，因此不建议将GST归一化用于芯片数据分析。

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).

问：如果我们在芯片上没有观察到抗GST信号，这是什么意思？是否没有足够的抗GST存在？还是蛋白质没有点在那个地方？

答：加入抗GST抗体只是为了方便网格对齐，因此只用低浓度的抗GST抗体（1:10000稀释），这会最大限度地减少抗GST抗体与正在研究的实际样本之间的竞争。因此，GST信号可能会在某些地方微弱甚至看不见。请注意，并非所有对照点都有GST标签。

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.

问：建议的实验流程中说明使用抗GST重新探测以建立参考网格，我应该在加入第二个探针之前去掉第一个探针吗？我可以同时用抗GST和我的一抗探测芯片，然后同时用两种二抗探测吗？这可以让我同时扫描两种抗体并节省处理时间。

答：不，在用抗GST重新探测之前不需要剥离一抗。只要不担心抗GST会干扰一抗，您可以将两种探针同时添加到芯片中，然后同时添加不同的二抗以检测结合的信号。

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.

问：在哪里可以获得高质量抗GST芯片染色的图像，以便与我们的结果进行比较？染色条件是什么？

答：每批点制芯片的GST图像都可用，并且可以从CDI网站资源页面下载。（http://cdi-lab.com/resources.shtml）。通常我们使用1:2000稀释的兔抗GST作为一抗，使用Alexa 555抗兔作为二抗。封闭缓冲液为含5% BSA的PBS-T，所用染色流程按用户手册规定来操作。

9.

Antibody Specificity and Cross-Reactivity Testing

抗体特异性和交叉反应测试

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.

问：在HuProt芯片上进行单克隆抗体（mAb）特异性测试需要什么浓度？

答：为了测试mAb特异性，建议使用0.1 – 1.0 μg/mL的浓度（取决于mAb亲和力）。

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.

问：我使用什么浓度的mAb来查看它是否与芯片上除抗原之外的其他蛋白质发生交叉反应？

答：要测试您的mAb与芯片上其他蛋白质的交叉反应，请将您的mAb浓度增加到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).

问：可以在每个芯片上测试多少个单克隆抗体的交叉反应性？芯片可以多次使用吗？竞争研究怎么样？

答：建议每个芯片仅用于交叉反应测试一次，因为点制的蛋白质芯片在扫描前的干燥过程中会变性。对于竞争研究，您可以同时将2个mAb加入到一个芯片中（每个mAb 0.1 – 1.0 μg/mL）。

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:

1.    Add the anti-GST antibodies and mAb sample to the array at the same time (not recommended).

2.    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.

问：对于抗体交叉反应测试，应该何时添加抗GST抗体进行网格对齐，应该使用哪个扫描波长进行抗GST染色的检测？

答：对于每个芯片使用一个mAb样品的分析，要检测抗GST染色，需使用与mAb染色不同的波长。有两种方法可以做到这一点：

<!--[if !supportLists]-->1.    <!--[endif]-->同时将抗GST抗体和mAb抗体与芯片孵育（不推荐）。

<!--[if !supportLists]-->2.    <!--[endif]-->为防止抗GST抗体与样品mAb竞争或与二抗发生交叉反应，首先用样品mAb进行检测，然后扫描芯片（大多数蛋白质斑点不可见）。接下来，将抗GST抗体加入到芯片上，然后将网格图案应用于mAb图像。由于使用低浓度的抗GST抗体，GST信号会很弱。

10.

Serum Profiling

血清分析

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.

问：我应该向CDI提供多少血清？

答：每份样品提供50 μL冷冻血清进行分析，最好干冰运输。由于反复冻融可能会影响检测结果，请将样品分成两等份，以便于需要稀释或重复检测。

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.

问：通常使用什么稀释度进行血清分析？

答：通常使用1:500或1:1000稀释来确保较低的背景。