Troubleshooting tips Membrane antibody arrays

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1 Troubleshooting tips Membrane antibody arrays Contents FAQ 1: FAQ 2: FAQ 3: FAQ 4: FAQ 5: FAQ 6: FAQ 7: FAQ 8: FAQ 9: Why are my positive control values different? Why is my background/ baseline so high? The background is uneven? Why are my signals weak or missing? Unequal intensities on duplicate spots Which steps can be done over night? How long can a solution keep fresh after dilution? Why are some of my spots white? How do I get spot intensities? FAQ 10: How do I do background correction? FAQ 11: How do I normalize the array data? FAQ 1: Why are my positive control values different? Separate pins print the spots and it is normal that the spots will have a slightly different size. This is called a Systemic error and will not affect the results since duplicate spots always should be averaged. The spots can be averaged in any combination, but it must be consistent. Variability of positive control is due to uneven incubation. This can be remedied as follows: 1. Washing (5 min wash buffer II) 2. HRP conjugate 3. Washing with Wash buffer I and II 4. Detection Discover more at abcam.com/technical 1

2 Sample of uneven incubation After optimization Sample 1 Sample 2 FAQ 2: Why is my background / baseline so high? If the background is high but even, adjusting of brightness & contrast of the image will help. Original image Adj. Brightness & Contrast NOTE: Such adjustments only affect image on screen, not densitometry High Background can be due to overexposure. We recommend always choosing at least two exposure times to make sure that a good image is collected for densitometry. If more than 75% of the spots are showing up and spots (positive control) are bleeding into each other, we call this an over-exposure. This can also indicate that the sample response is too low and it has to be used more concentrated. Discover more at abcam.com/technical 2

3 When crude lysates are used as sample, high background is often seen due to the high amount and number of proteins in lysates. We recommend to further dilute the lysate to reduce the background. 1 st time 500µg /ml 2 nd time 250µg /ml Summary 1. Overexposure - Decrease exposure time 2. Membranes were dried out during experiment - Completely cover membranes with solution during experiment 3. Sample is too concentrated - Use more dilute sample 4. High concentration of HRP - Make sure the correct dilution and amount of HRP 5. Repeat wash step overnight, then repeat detection - 1. Washing (5 min wash buffer II) 2. HRP conjugate 3. Washing with Wash buffer I and II 4. Detection FAQ 3: The background is uneven? 1. Do all incubation steps on a rocker or shaker 2. Avoid bubble formation during incubation 3. Make sure the samples volume is correct and do not use too little sample 4. Take care that the membrane does not dry out during incubation 5. Dilute the sample further if concentration is high or sample is very viscous Discover more at abcam.com/technical 3

4 FAQ 4: Why are my signals weak or missing? The key determinant of cause is discerned using the positive controls: 1. If the detection works, positive signals will be visible. 2. If positive signals are weak, then it's procedural or reagents. Please make sure no steps in the protocol have been omitted. Repeat incubation with conjugated HRP and repeat detection. Detect chemiluminescence immediately. 3. If positive signals are strong, sample is below detectable limits. We suggest to use a higher concentrated samples and a positive control sample. A longer incubation time of the sample (over night 4 C) might also help. Customer Image Repeat HRP FAQ 5: Unequal intensities on duplicate spots If same spots are affected on multiple membranes this is a System Error and it will not affect the results since a ratio of the spots will be used to calculate any intensities. FAQ 6: Which steps can be done over night? Blocking Incubations Sample Biotinylated antibody Streptavidin conjugated HRP Wash Steps Wash Buffer II Make sure to cover the sample and avoid evaporation. Incubation at 4 C overnight. FAQ 7: How long can a solution keep fresh after dilution? We recommend to dilute all reagents immediately before using them. In general the working solutions can be kept at 4 C for up to 2-3 days. Discover more at abcam.com/technical 4

5 FAQ 8: Why are some of my spots white? Cause 1: high concentration of HRP Mix well the HRP streptavidin before use. HRP may precipitate during storage. High concentration of HRP will cause white spot. Cause 2: high concentration of samples Reduce the amount of samples. We recommend to dilute samples at least 10 fold using blocking buffer Cause 3: high concentration of endogenous HRP or H 2 O 2 Dilute samples with block buffer. Decreasing of the exposure time may also reduce white spot. FAQ 9: How do I get spot intensities? Any densitometry software suitable for 2D gels is sufficient. Best to use a gel documentation system software is included. ImageJ is available for download for free from NIH FAQ 10: How do I do background correction? Average the negative control spots Subtract this value from every spot on the array You may want to run a negative control array and subtract from all other arrays FAQ 11: How do I normalize the array data? Pick any two spots on the membrane that appear in all membrane you want to normalise The ratio of these spots will be the same on all membranes Pick the spot of interest and measure with densitometry : Example of calculation for the normalized array of TNFα shown on the attached image. After background subtraction, use the formula: ) = ITNFα test x IPos Ref IPos test ) = ) = 2000 x Legend: ): normalized array data for TNFα ITNFα test : mean signal density for TNFα on the membrane of interest (e.g 2000) IPos Ref : mean signal density of the positive controls on the reference array (e.g 4500) IPos test : mean signal density of the positive controls on the membrane of interest (e.g 6000) After normalization, you can compare the relative expression levels against IPos Ref that is set up as 1. The ratio for TNFα is here: 1500 / 4500 = 0.3 Discover more at abcam.com/technical 5