Western Blotting Study Guidebook Chapter 1

3 WESTERN BLOTTING TIPS AND GUIDELINES WESTERN BLOTTING TIPS AND GUIDELINES 3.1. Tips for Successful Western Blot Transfer Whether you choose to do a wet or dry transfer, there are many steps you can take to maximize transfer efficiency. Wet Transfer Benefits of Wet Transfer  sing a wet transfer apparatus, high intensity U (1-2 hour) or lower intensity (overnight) transfers can be performed. This allows for optimizing transfer conditions for each individual protein. Transfer times can be shortened to prevent transfer of low molecular weight proteins through the membrane while longer transfer times can be used to promote complete migration of high molecular weight proteins out of the gel. Transfer “stack” Tank Cassette Drawbacks to Wet Transfer  The transfer process generates heat, which can decrease the resistance of the transfer buffer resulting in inconsistent transfer across the gel. High heat can also result in breakdown of the gel itself. To prevent heating, transfer buffer should be pre-chilled prior to use. In addition, the transfer buffer should be kept cold during transfer. Long transfers are often performed in a cold room to aid in keeping the buffer cool. Additionally, selfcontained ice blocks can be placed within the tank and are usually supplied with the apparatus. Alternatively, external-cooling mechanisms can be used to control heating. Advantages Disadvantages • Flexibility: • Transfer conditions are easily adjusted • Multiple transfer buffer options enable more ways to optimize transfer • Supports transfer of a broad molecular weight range at one time • Compatible with extended transfer times for large molecular-weight proteins • Can be used for quantitative Westerns • H  eating of buffer can interfere with transfer • Cooling mechanism and/or cold room space required during transfer • Large volumes of transfer buffer are required Semi-Dry Transfer Benefits of Semi-Dry Transfer  Semi-dry transfers are fast and easy and require little buffer. Transfer of more difficult to transfer proteins can be optimized by using a discontinuous buffer system, a feature unique to semi-dry transfer systems. In a discontinuous buffer system, the filter paper placed on the anode side of the stack is soaked in a different buffer from the filter paper placed on the cathode side of the stack. This can increase migration of the protein out of the gel while also promoting better retention on the membrane. Apparatus Cover Electrode Plate + + Transfer “stack” Electrode Plate – – Apparatus Bottom Power Cord Drawbacks to Semi-Dry Transfer Semi-dry transfer systems have less flexibility and it can be difficult to transfer both high and low molecular weight proteins. Low molecular weight proteins can transfer through the membrane due to the high intensity blotting conditions while high molecular weight proteins may not efficiently transfer out of the gel due to decreased transfer times. However, transfer of high molecular weigh proteins can be improved by using the discontinuous buffer system. Transfer times cannot be extended when using semi-dry transfer, as there is limited buffering capacity. In addition, the gel can dry out if insufficient buffer is used. Advantages Disadvantages • Transfer is rapid • Can use discontinuous buffer system to optimize transfer of proteins • Little buffer is required • Easy to set up • Good for performing large numbers of blots analyzing the same protein • H  igh intensity field strength may cause low molecular weight proteins to migrate through membrane • D  ifficulty in transferring high (>120 kDa) molecular weight proteins • N  ot recommended for quantitative Westerns • Gel can dry out Tips for Successful Transfers • Take  care when preparing transfer buffers; small inconsistencies can affect transfer • Use high quality, reagent grade methanol when preparing buffers; impurities in methanol can decrease transfer efficiency • Never dilute the transfer buffer • Do not adjust pH of the transfer buffer • For optimum transfer, do not reuse transfer buffer • Make sure all equipment is clean • Use a thinner gel (0.5-0.75 mm thickness) • If using a PVDF membrane, pre-wet the membrane in 100% methanol prior to equilibration in transfer buffer • Equilibrate transfer pads, filter paper, membrane and gel for at least 15 minutes in buffer(s) • Remove all air bubbles and creases between each layer in the stack • Ensure the stack is firmly held in place and pressure is applied evenly over the entire surface of the stack • Ensure that electrodes are connected and free of debris and damage (plate electrodes) • Make sure the current is applied correctly and proteins will migrate towards the membrane • Use pre-stained molecular weight standards to help monitor transfer • Stain the blot with a reversible total protein stain (e.g. Ponceau S) to check quality of transfer • For difficult to transfer proteins, adjust methanol and SDS concentrations. SDS promotes migration of proteins out of the gel but can inhibit membrane binding. Methanol increases retention of proteins on the membrane but can hinder migration out of the gel. WESTERN BLOTTING TIPS AND GUIDELINES Wet Transfer Buffers  Towbin  Transfer Buffer • Standard wet transfer buffer • SDS (0.025-0.1%) can be added to facilitate transfer of proteins 25 mM Tris, pH 8.3 192 mM glycine 20% methanol +/- SDS CAPS (3-[cyclohexylamino]-1 propane sulfonic acid) Buffer • For blotting basic proteins • For blotting prior to N-terminal sequencing 10 mM CAPS, pH 11 10% methanol Dunn Carbonate Buffer • For higher efficiency transfer of basic proteins • Can be used to enhance ability of antibodies to recognize some antigenic sites 10 mM NaHCO3 3mM Na2CO3, pH 9.9 20% methanol Semi-Dry Transfer Buffers Bjerrum Schafer-Nielsen Buffers • Standard semi-dry transfer buffer based on Towbin 48 mM Tris, pH 9.2 39 mM glycine 20% methanol Discontinuous Tris-CAPS Buffer System • Uses two different buffers to enhance transfer of proteins • The filter paper assembled on the membrane side (anode) of the blot contains methanol • The filter paper on the gel side (cathode) of the blot contains SDS 60 mM Tris, pH 9.6 40 mM CAPS + either 15% methanol or 0.1% SDS 3.2. How to Improve Your Chemiluminescent Western Blots Chemiluminescent Westerns are popular assays for assessing protein expression. In this indirect detection method, chemiluminescent substrates emit light when reacted with an antibody conjugated to an enzyme. The emitted light is captured and archived on x-ray film (traditional), or through digital imaging. Chemiluminescent Western blotting is a highly sensitive assay and can detect femtograms of protein. Although it is only semi-quantitative, it is useful for detecting the presence or absence of a protein. For example, chemiluminescence can be used to detect the induction of exogenous protein expression, to confirm and follow purification of a known protein, or for verification of antibodies during production. Substrate HRP Secondary Antibody Primary Antibody Protein Sample Chemiluminescent Westerns can be difficult to perform. The ultimate goal is to obtain a blot with a high signal-tonoise ratio. However, chemiluminescent Westerns can be plagued with high background, either in the form of an overall background that masks the signal from the protein of interest, or as bright dots and speckles and/or splotches scattered randomly over the blot. The increase in background noise can arise from a variety of factors. It can also be difficult to obtain a strong signal from the protein of interest. Tips for Improving Chemiluminescent Westerns • Optimize the amount of protein to load on the gel. In general, 20-40 μg of total protein can be loaded without overloading the well. However, the total amount of protein should be optimized for each protein:antibody pair. Ideally, enough protein should be loaded to allow for easy capture of the signal without experiencing saturation. • Choose the correct membrane. Nitrocellulose and PVDF membranes are commonly used, and each has advantages and disadvantages. • Keep everything clean. Prevent background by thoroughly cleaning all equipment and trays prior to use. Only handle the gel and membrane with gloved hands. Keep trays covered during incubations. •  Ensure that all buffers are well mixed. Particulates in blocking and antibody incubation buffers will stick to the membrane and cause high background. Buffers can be filtered prior to use. • If experiencing high background, use a larger volume of washing buffer and increase number and duration of washes. •  Try different blocking buffers. Some antibodies react with proteins in blocking buffers, causing a high background. Alternatively, some blocking buffers can mask the protein of interest, preventing detection. Nonfat dry milk and bovine serum albumin are the two most common protein blockers containing multiple proteins. Blocking buffers containing one protein can also be used. Protein-free buffers can be used when the primary antibody reacts with protein components in the buffer. • Titrate both primary and secondary antibodies. Use a dot blot and checkerboard titration to determine the optimum primary and secondary antibody concentrations. • Never dilute a horseradish peroxidase-conjugated secondary antibody in buffer with sodium azide. Sodium azide inhibits HRP activity. • Use enough substrate. Make sure the blot is coated entirely with substrate to prevent local concentration differences. • Try different substrates to increase sensitivity and signal duration. Different substrates are available with differing sensitivities for detecting high to moderate versus low abundance proteins. Different substrates also have different reaction rates and emit light for different durations of time. This can affect your ability to capture multiple exposures. • The substrate may need to be equilibrated to room temperature before use to increase the enzyme activity. • Use a digital imager rather than film. Digital imagers increase the linear dynamic range allowing easier detection of low abundance proteins while limiting saturation when detecting high abundance proteins. WESTERN BLOTTING TIPS AND GUIDELINES 3.3. How to Improve Your Fluorescent Western Blots Fluorescent Western blots are the gold standard for quantitative Westerns. They are ideal for detecting multiple proteins simultaneously (multi-plexing), allowing in-lane normalization and detection of your protein of interest and loading control at the same time. In addition, post-translational modifications can be studied and quantitated easily. Secondary Antibody Secondary Antibody Primary Antibody Protein A Primary Antibody Protein B In fluorescent Western blotting, the secondary antibody is directly conjugated to a dye, which is excited by light. The emitted light is detected by a digital imager and digitized for data analysis. Multiple proteins can be detected simultaneously by using secondary antibodies conjugated to different dyes with non-overlapping spectral emissions. Although similar to chemiluminescent Westerns, there are additional factors that must be taken into consideration when performing fluorescent Westerns: • Titrate both primary and secondary antibodies. Use a dot blot and checkerboard titration to determine the optimum primary and secondary antibody concentrations. • When multi-plexing, optimize detection of each target separately prior to simultaneous detection. • Primary antibodies may need to be increased 2-5x compared to concentrations used in chemiluminescent Westerns. • Secondary antibodies may also need to be increased (1:5000 is a recommended starting dilution). •  Use a PVDF membrane with low autofluorescence. Nitrocellulose and some PVDF membranes can autofluoresce causing high background. • Avoid inks and dyes that can fluoresce. Use a pencil to mark the blot. Common dyes such as bromophenol blue and Coomassie autofluoresce. • Keep everything clean. Prevent background by thoroughly cleaning all equipment and trays prior to use. Only handle the gel and membrane with gloved hands. Use powder-free gloves. Keep trays covered during incubations. • If you are using fluorescent molecular weight markers, skip a lane before loading samples. This will prevent the signal from the molecular weight markers from bleeding into sample lanes. • Work with fluorescent antibodies on the bench top, but store stocks in the dark. • When multiplexing, use primary antibodies made in different species and secondary antibodies that are highly cross-adsorbed to prevent cross recognition. • Avoid spectral overlap when multiplexing. Choose fluorophores that have optically distinct spectra. • To increase the specific signal, always detect the strongest target in the blue channel, the middle in the green channel and the weakest in the red channel. • When archiving blots, store them in the dark. 3.4. Transition from Chemiluminescent to Fluorescent Western Blots Bring Fluorescent Western Blotting to Your Lab. It’s Easier Than You Think. Chemiluminescence is the most familiar method of detection for Western blotting and offers great sensitivity. However, many scientific questions and experimental designs require the additional information provided by fluorescent Western blotting; this includes precise quantitation and visualization of similarly sized proteins within the same sample. Once you make the decision to move to fluorescent Western blotting, what comes next? First, you can check if fluorescent Western blotting is right for your experiment using the flow chart in the figure below. Then, refer to the tips and advice provided in this document to get started with your first fluorescent Western blot. Do you have access to a digital imager? No Yes Do you have multiple proteins to detect on the same blot? No Yes Are they similar sizes? No Do you anticipate a very low level of protein? No Chemiluminescence Yes Do you need precise quantitation? Yes No Either Yes Fluorescence Chemiluminescent or Fluorescent Western Blotting. Before You Run the Gel Dilute Ladder Some pre-stained protein ladders fluoresce strongly and can interfere with detection of proteins expressed at a low level. Try diluting the ladder 1 to 10 before loading. Optimize Sample Concentration As with any new assay, quality results are dependent on first optimizing your assay for the samples to be used. Sample load volume may need to be adjusted for the best possible results. When first switching from chemiluminescent to fluorescent Westerns, try a slightly higher sample load volume to confirm that your assay is working. Once you have a positive result, you can lower the sample load volume and push for more sensitive results. Make considerations for the protein ladder, sample concentrations, and loading dye. WESTERN BLOTTING AND GUIDELINES WESTERN BLOTTING TIPSTIPS AND GUIDELINES While Running the Gel Be Wary of Loading Dye 1 2 3 4 5 6 7 8 9 10 Many loading dyes can fluoresce. To prevent this from interfering with the signal on a fluorescent blot, run the dye front off the gel or cut it off before transfer. Transferring to the Membranel Before setting up the transfer, make sure to trim off the loading dye if possible to prevent it from interfering with your data. PVDF Membranes For the best signal-to-noise ratio, use a membrane with minimal autofluorescence such as Azure’s low fluorescence PVDF membranes. Membrane Handling Any contamination to the membrane will be obvious in a fluorescent Western blot. Always use forceps when handling membranes. Products • Pre Cut PVDF (AC2108 – AC2109) Use PVDF membranes and handle only with forceps. Probing the Membrane Blocking As with chemiluminescent blots, it is important to completely block the membrane to avoid nonspecific binding of antibodies and to reduce background. Azure Protein Free Blocking Buffer and Azure Fluorescent Blot Blocking Buffer are formulated to not only reduce background, but also to stabilize the fluorescent dyes of AzureSpectra secondary antibodies for enhanced signal detection. Detector Molecule Secondary Antibody Primary Antibody Optimize Antibodies Antibody concentration may need to be optimized and adjusted for the best possible results. When first switching from chemiluminescent to fluorescent Westerns, use the manufacturer’s recommendations for antibody dilutions. Incubation Trays Target Membrane Indirect Secondaryof antibody is labeled. Diagram the antigen-antibody and antibody-antibody interactions in Western blotting detection. Fluorescent secondaries can become quenched when exposed to bright light for long periods of time. When incubating your sample with light sensitive antibodies, cover your blot with an opaque material (such as with Azure’s opaque incubation trays) to protect from quenching. Products • • • • Azure Protein Free Blot Blocking Buffer (AC2112) Azure Fluorescent Blot Blocking Buffer (AC2190) Fluorescent secondary antibodies (AC2128 – AC2139, AC2156 – AC2171) Opaque Incubation Trays (AC2120 – AC2123) Use opaque containers to protect the light-sensitive fluorophores during incubation. Secon Washing Stringent Wash Because the fluorescent dyes can adhere to the membrane, washing is extremely important to reduce background. Make sure to use a stringent wash, especially when working with near-infrared fluorophores. Azure Fluorescent Blot Wash Buffer is specially formulated for use with fluorescent Western blots. Wash Volume The volume and duration of washing is also important to rid the membrane of any free dye and antibody. We recommend two quick rinses in 25mL wash buffer followed by three 5 minute washes in 25mL wash buffer. To prevent background signals and get the clearest data, follow the recommended steps such as thorough washes after antibody incubation. Final Rinse Any detergent in the wash buffer can also fluoresce, so rinsing in PBS or TBS after the final washing step is essential to lower background signal. Products • • Azure Blot Washing Buffer (AC2113) Azure Fluorescent Blot Washing Buffer (AC2145) Imaging Reduce Background Contamination When many people use the same imager, the insides and trays can become contaminated. Background Quenching Sheets absorb background fluorescence to improve signal-to-noise ratios. Products • Quenching sheets (AC2144, AC2147)  Want some help getting started? A demo kit is a great way to try out fluorescent Western blotting in your own lab with your own samples and primary antibodies. It contains everything you need to perform single color fluorescent Western blotting: • PVDF Membrane • Fluorescent Blot Wash Buffer • Fluorescent Blot Blocking Buffer • Secondary Antibody • 2 Quenching Sheets AzureSpectra Demo Kits • Goat α-Mouse 700 (AC2172) • Goat α-Rabbit 700 (AC2173) • Goat α-Mouse 800 (AC2174) • Goat α-Rabbit 800 (AC2175) • Goat α-Mouse 650 (AC2176) • Goat α-Rabbit 650 (AC2177) • Goat α-Mouse 550 (AC2178) • Goat α-Rabbit 550 (AC2179) To prevent background fluorescence from interfering with your signal, use Azure’s quenching sheets. WESTERN BLOTTING AND GUIDELINES WESTERN BLOTTING TIPSTIPS AND GUIDELINES 3.5. Western Blot Normalization Quantitative Westerns: What is the Best Way to Normalize Your Western blot? Far  from being an “is-it-there-or-not” technique, modern digital detection instruments can make Western blotting reproducible and quantitative. By working within the linear dynamic range of your detection method and normalizing the data to control for variations in protein load and membrane transfer, you can get truly quantitative results. But what is the best way to normalize a Western blot? In the past, the gold standard normalization method was to use a housekeeping protein based on the assumption that the levels of these proteins are fairly consistent across experimental conditions and cell lines. However more recent studies have shown that this assumption is not always true1,2 leading to inaccurate measurements of relative protein abundance. Instead, quantitative Western blotting experts1,2 and the journals they publish in4 are recommending a new gold standard for normalization—normalizing to total protein detected in each lane, preferably by staining on the membrane. Western Blot Normalization: Housekeeping Protein vs Total Protein Normalization method Housekeeping protein Total protein Benefits • Familiar, commonly used • • • • Challenges • N  arrow linear dynamic range • Abundance can vary with experimental conditions • Abundance may not be consistent between sample types • High variability • Must ensure housekeeping protein physically resolved from protein of interest on gel • M  ust ensure total protein stain used is compatible with antibody binding and detection method Large linear dynamic range Low variability Consistant across sample types No change with experimental conditions Using Housekeeping Proteins for Normalization The most significant drawback of using housekeeping proteins is that their levels may not be consistent across samples and conditions.1,2 It is possible to use a housekeeping protein for normalization, but you must first spend the time and effort to validate your choice, and may need to examine multiple potential standards before you find one that is truly expressed at the same level across all of your samples and does not change across your experimental conditions. A  second significant challenge associated with housekeeping proteins is their high abundance.1,3 If the housekeeping protein is present at a very high level in your sample, this limits the amount of sample you can load on the gel because you will need to keep the housekeeping protein within the linear range of detection and not saturate the signal for the housekeeping protein. This is particularly problematic if the protein of interest is not similarly highly expressed because the two proteins will not be within the same linear range of detection.2,3 A third challenge to consider if you’re doing multiplex Western blots—such as comparing phosphorylated and non-phosphorylated forms of the same protein—is the complexity of generating primary and secondary antibodies from non-overlapping species.  Finally, it is always possible that detecting the housekeeping protein could interfere with detection of the protein of interest.1 Ideally, the housekeeping protein should be a different size than the protein of interest so the two proteins are spatially resolved on the blot. This becomes increasingly difficult when an experiment examines multiple proteins of interest on the same blot. Using Total Protein Staining for Normalization With total protein normalization, instead of trying to find a protein that can represent the total amount of sample that transferred to the membrane, total protein is measured on the membrane directly and this value is used as the denominator when normalizing.1-4 Many total protein stains are available that can be used to stain gels and membranes.1 Total protein stains provide a larger dynamic range and demonstrate lower variability and cleaner data than housekeeping proteins.1,2 Traditional Western Blot Workflow Western Blot Workflow with AzureRed TPS Separate proteins on gel Separate proteins on gel Transfer proteins to membrane Transfer protiens to membrane Stain for total protein with AzureRed (30-minute protocol) Block membrane Block membrane Total protein normalization can be much faster than using a housekeeping protein, especially for chemiluminescent blots Finish Western blot protocol Finish Western blot protocol because the staining step takes less time that stripping and reprobing the blot. Ideally, total protein staining is conducted on the membrane, either before or after immunodetection.2 With some stains such as AzureRed Fluorescent Total Protein Stain, it is possible to stain the blot before immunodetection and then to image total protein simultaneously with the protein(s) of interest. With this simplest of workflows, images for the protein(s) of interest and total protein are automatically aligned, avoiding the need resize and align images captured at different times. The analysis workflow after image capture is essentially unchanged compared to using a housekeeping protein; the signal density for the entire lane or a large portion of the lane is used for normalization instead of the density for a single band. Staining the membrane with a total protein stain provides an added quality control benefit, allowing verification that membrane transfer was complete and free of artifacts. Looking for More Information About Western Blot Normalization? Please see the Western Blot Normalization application note to learn more about the different methods of Western blot normalization and to help decide which normalization approach is best for your blot. Read more about using AzureRed Fluorescent Total Protein Stain for total protein normalization of fluorescent Western blots in the Accurate Western Blot Normalization With AzureRed Fluorescent Protein Stain application note. References 1.  Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. Proteomics. 2017;17:1600189. 2. Thacker JS et al. Total protein or high-abundance protein: which offers the best loading control for Western blotting? Anal Biochem. 2016;496:76-78. 3. McDonough AA et al. Considerations when quantitating protein abundance by immunoblot. Am J Cell Physiol. 2015;308(6):C426-C433. 4. Fosang AJ, Colbran RJ. Transparency is the key to quality. J Biol Chem. 2015;209(50):29692-29694. WESTERN BLOTTING TIPS AND GUIDELINES 3.6. Images Used: 2015-1228-164847 2016-0105-153125 The Problem: Troubleshooting Images Used: 2015-1228-164847 2016-0105-153125 Membrane Not Fully Stripped The Problem: Membrane Not ImageFully Stripped The problem The fix Membrane not fully stripped • E  nsure adequate buffer volume and stripping time The Fix: Images Used: 2015-1222-163006 Images Used: 2015-1230-163715 2015-1222-163006 2016-0105-153125 2015-1230-163715 2016-0105-153125 The Problem: The Problem: Insufficient The Fix: • Ensure adequate buffer volume or Incorrect The Fix: The Problem: and stripping time Insufficient or Incorrect to Stripping Wash Prior Insufficient Insufficient or • or R  inseIncorrect blots with ultra Wash Prior to Stripping Resulting images The Fix: • Ensure adequate buffer volume Images Used: and stripping time 2015-1222-1630 The Fix: 2015-1230-1637 2016-0105-1531 Prior to Stripping Wash prior to stripping minutes before stripping incorrect wash pure water for at least 5 • D  o not wash with wash buffer • Rinse blots with ultra Not washed before stripping Washed purewith water for at least 5 • Rinse blots ultra Imagesstripping Used: minutes before with wash pure water for at least 5 2015-1209-091112 2016-0107-101420 • Do not wash with buffer before minutes before stripping wash The Problem: bufferwith wash Washed with wash buffer before•stripping Not washed before stripping stripping Do not wash The Problem: Marker Too Bright Thebuffer Fix: Marker Washed with wash buffer before stripping Not washed before stripping Too Bright Images Used: 2015-1209-091 2016-0107-101 The •Fix: Rinse blots with ultra • C  over the marker before imaging • A  llow blot to dry before imaging D • • Cover the marker before  ilute marker before Washed with wash buffer before stripping Not washed imagingbefore stripping loading Marker too bright The Problem: • Allow blot to dry before imaging • Dilute marker before loading The Problem: Bubbles in Transfer Marker Too Bright Bubbles Thein transfer Fix: Tips for transferring proteins from gel to membrane 1. Optimize voltage and duration of transfer for each device 2. Remove air bubbles before transfer 3. Ensure transfer stack has a tight fit Transfer stack was not tight: Replace sponge pads Air bubbles: Ensure bubbles are removed or degas transfer buffer Images Used: 2016-0104-161425 2016-0107-095429 2016-0107-101420 • P  ress and smooth filter paper after covering membrane to remove bubbles • E  nsure adequate transfer buffer is present in cartridge • Press and smooth filter paper after covering membrane to Contamination: Clean remove bubbles Finger prints: Always use equipment and replace forceps to transfer handle • Ensure adequate sponge pads membrane buffer is present in cartridge pure water for at least 5 minutes before stripping • Do not wash with wash buffer Images Used: 2015-1209-091 before 2016-0107-101 • Cover the marker imaging • AllowFix: blot to dry before The imaging • Dilute marker before loading • Cover the marker before imaging • Allow blot to dry before imaging • Dilute marker before loading Images Used: 2016-0104-164834 2016-0111-171150 2016-0107-150652 Images Used: 2016-0104-164834 2016-0111-171150 2016-0107-150652 The Problem: InsufficientImage or Uneven Transfer Pressure The Problem: Insufficient or Uneven The fix The Fix: Transfer Pressure The problem Insufficient or uneven transfer pressure • T  ransfer set up should be very tight • Place gel and membrane in the middle of the casette • Transfer set up should be very tight • Place gel and membrane in the middle The Problem: The Problem: Resulting images The Fix: • Transfer set up should be very tight Images Used: • Place gel and membrane in 2015-1230-164415 2016-0106-162523 the middle Images Used: 2015-1230-164415 2016-0106-162523 2015-1214-151723 2015-1214-151723 Antibody Cross-reactvity Antibody The Fix:Cross-reactvity e Problem: or Quality • A  void antibodies from species that are too “close” (ex. rat and mouse) • Avoid secondaries for the animal in which another • Avoid antibodies from species was that created secondary are too “close” (ex. ratIfand mouse) (ie. using one goat Images Used: • Avoid secondariesanti-rabbit, for the animal in using a avoid 2015-1209-125309 which another secondary was donkey anti-goat in the 2015-1217-173919 created (ie. If using one goat antirabbit, avoid usingsame a donkey anti2015-1216-151617 assay) goat in the same assay) • antibodies Block in between • Block in between that might cross react antibodies that might cross-react The Fix: Antibody cross-reactvity The Fix: AntibodyThe Problem: Poor qualityAntibody • Screen antibodies Poor Quality antibody • Avoid antibodies from species that are too “close” (ex. rat and mouse) • Avoid secondaries for the animal in which another secondary wasImages Used: created (ie. If using one goat anti2015-1209-125309 rabbit, avoid using a donkey anti2015-1217-173919 goat in the same assay) 2015-1216-151617 • Block in between antibodies that might cross react The Fix: • Other causes: • Non-specific binding • Too much antibody • Insufficient blocking Images Used: 2016-0106-161352 2016-0107-094635 The Problem: High Background • Screen Theantibodies Problem: The Fix: High Background • Other Causes: Non-Specific HighBinding background • Use low-fluorescence membrane • Too much•antibody Use correct washing and wash buffer • Insufficient protocol blocking Images Used: 2016-0106-161352 2016-0107-094635 The Fix: antibodies • Screen • Other Causes: Non-Specific Binding • Too much antibody • A  fter washing rinse with • Insufficient blocking water • Do not use excessive amounts of antibody Ensure samples are free of cellular debris and are not viscous Load samples slowly and make sure not to overload the wells • Dry the membrane before • The usual: membrane, blocking, Optimize the amount of protein loaded washing, rinsing, antibody load imaging blocking, • The usual: membrane, Tips for preparing samples and loading gels 1. 2. 3. Cellular debris in samples: Sonicate and filter 250 150 100 75 50 37 25 20 15 10 Cell SV1 SV2 SV3 S1 S2 S3 S4 S5 washing, rinsing, antibody load Cellular• debris in membrane • Sonicate and filter Dry the before imaging DNA in samples: Shear samples with syringe if in sample buffer or add DNase samples • F  or DNA in samples, shear samples with syringe or Transferrin add DNase Actin GAPDH • Dry the membrane before imaging