Optiblot. Western Blot Reagents and Troubleshooting Tips. Discover more at abcam.com/optiblot

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1 432_12_GM Optiblot booklet02_layout 1 28/08/ :14 Page 1 Optiblot Western Blot Reagents and Troubleshooting Tips

2 High quality protein electrophoresis and western blot reagents Our Optiblot range of products for protein electrophoresis and western blotting is designed to save you time and ensure you can confidently and consistently produce superior results. The Optiblot range includes electrophoresis gels, buffers, accessories and reagent bundles which offer a number of features which your lab can benefit from: Optiblot electrophoresis gels Gel cassette tank compatibility: Optiblot gels are compatible with commonly available tank systems Tank system 10 x 10cm Optiblot gel 8 x 10cm Optiblot gel Bio-Rad Mini-PROTEAN No Yes Invitrogen XCell II Yes No Invitrogen XCell Surelock Yes No Thermo Scientific Owl P82 Yes No CBS Scientific Quadra Combo Yes Yes Easy to use Coloured wells for simplified loading High capacity wells and no comb to remove Up to 10x stronger than common gels No more torn gels Long shelf life No more wasted gels with 12 month shelf life SDS Gels Product List: Product name Cassette Size Pack Size Product code Optiblot SDS Gel 10% (12 wells) 10 x 10cm 10 gels ab Optiblot SDS Gel 12% (12 wells) 10 x 10cm 10 gels ab Optiblot SDS Gel 4-8% (12 wells) 10 x 10cm 10 gels ab Optiblot SDS Gel 4-20% (12 wells) 10 x 10cm 10 gels ab Optiblot SDS Gel 10% (12 wells) 8 x 10cm 10 gels ab Optiblot SDS Gel 12% (12 wells) 8 x 10cm 10 gels ab Optiblot SDS Gel 4-8% (12 wells) 8 x 10cm 10 gels ab Optiblot SDS Gel 4-20% (12 wells) 8 x 10cm 10 gels ab Easy to open No need for spatulas or knives Compatible with most tank systems For a complete product listing visit: Buffers and accessories Optiblot accessories include reagents for sample preparation, electrophoresis and membrane transfer. Choose from single concentration gels (8%, 10%, 12% and 16%) or gradient gels (4-8%, 4-12% and 4-20%) with a continually expanding range. SDS-PAGE Gels available in 12 well and 17 well format and in cassette sizes of 10 x 10cm or 8 x 10cm for respective tank sizes (including XCell and Mini-PROTEAN gel tanks). Sample Preparation: Sample preparation kit (ab133414): 10 minute protocol - for fast and efficient sample concentration and removal of buffer components that may interfere with electrophoresis (GuHCl, Urea, ammonium sulphate etc). DTT Reducer (ab119199): Add during sample preparation in order to perform reducing SDS-PAGE. Bradford Reagent (ab119216): Simple and rapid Coomassie based method for protein quantitation. Detergent compatible (with up to 1% detergent) Can tolerate UV absorbing chemicals and reducing reagents present in a protein sample (unlike BCA or UV methods). 1 2

3 Electrophoresis: Optiblot buffers are specifically designed for use with Optiblot gels to provide optimal running and transfer conditions. Reducing Buffer (ab119195): Reducing Tris-MOPS buffer similar to conventional MES buffer systems: optimal separation of low molecular weight proteins. SDS Run buffer (ab119197): Non-reducing Tris-Tricine buffer similar to a conventional MOPS buffer: increased separation of higher molecular weight proteins. Antioxidant (ab119200): Recommended when running reduced samples - the antioxidant runs ahead of the dye ensuring that the environment the protein is migrating through is fully reduced. This prevents oxidation of the sample and the resulting loss in resolution. Gel Drying Solution (ab119201): To dry Optiblot precast gels for long term storage. Optiblot Blue (ab119211): For sensitive and safe staining of protein bands in a single step. Visualize protein bands in 15 minutes Transfer: Optiblot TGS blot stock buffer (ab119198): Designed for efficient transfer of proteins from Optiblot precast gels. Transfer membranes: For convenient blotting, choose from a range of quality controlled nitrocellulose and PVDF membranes: Product name Membrane size Product code Low Fluorescence Western Membrane (PVDF) 9x7 cm (10 sheets) ab Nitrocellulose Transfer Membrane µm 8x10 cm (10 sheets) ab Nitrocellulose Transfer Membrane µm 8x10 cm (10 sheets) ab Protein ladders: For producing sharp and visible bands choose Optiblot or Prism pre-stained multi-colored protein ladders. These pre-stained molecular weight markers can be used for estimating protein size as well as confirming western blot transfer efficiency. Product name Range Size/description Product code Optiblot Pre-stained Marker kda 600µl ab Prism Protein Ladder kda 500µl ab Prism Ultra Protein Ladder kda 500µl ab Prism Ultra Protein Ladder kda 500µl ab Prism Ultra Protein Ladder kda 500µl ab For further information about Prism protein ladders visit: No need to wash, microwave or destain Rapid gel stain - visualize protein bands in 15 minutes (no need to wash, microwave or destain) Easily identify your protein with Prism ladders 3 4

4 Buffers and accessories product list: Product name Size/description Product code Sample preparation SDS-PAGE Sample Preparation Kit 10 or 20 tests ab DTT Reducer (10X) 1ml ab Optiblot Bradford Reagent 500ml or 1000ml ab Electrophoresis Reducing SDS Run Buffer 500ml or 5L ab SDS Run Buffer (20X) 500ml or 5L ab Antioxidant (800X) 10ml ab Gel Drying Solution 1000ml ab Optiblot Blue 1000ml ab Optiblot Pre-stained Ladder ( kda) 600µl ab Prism Pre-stained Ladder ( kda) 500µl ab Prism Ultra Pre-stained Marker ( kda) 500µl ab Prism Ultra Pre-stained Marker ( kda) 500µl ab Prism Ultra Pre-stained Marker ( kda) 500µl ab Transfer Optiblot Gel Drying Solution 1000ml ab Low Fluorescence Western Membrane (PVDF) 9x7 cm (10 sheets) ab Nitrocellulose Transfer Membrane µm 8x10 cm (10 sheets) ab Nitrocellulose Transfer Membrane µm 8x10 cm (10 sheets) ab Run buffer recipes available to download at ECL kits: Optiblot ECL Detect kits give you a strong signal with low background whilst using less antibody. Optiblot ECL Detect Kit (23pg-187ng) (ab133406) Load less sample conserve precious sample Detect low pg amounts of protein for detecting low abundance proteins Use up to ten-times less antibody conserve precious antibodies Long lasting signal carry out multiple exposures at your convenience without signal decay Stable signal carry out longer exposures to detect low abundance proteins Optiblot ECL Max Detect Kit (4.6pg-4.7ng) (ab133408) High sensitivity (detect attomoles of protein) ideal for detecting low abundance proteins Long lasting signal carry out exposures at your convenience Quantitative broad linear dynamic range of signal with respect to protein amount Broad linear range (above 3 orders of magnitude) detect low and high abundance proteins in the same exposure Versatility optimized for CCD imaging and compatible with film detection Flexible - image chemiluminescence by CCD or film, image chemifluorescence with laser based fluorescence imaging systems Optiblot ECL Ultra Detect Kit (1.2pg-2ng) (ab133409) Highest sensitivity (detect attomoles of protein) ideal for detecting low abundance proteins using small amounts of antibody Quantitative broad linear dynamic range of signal with respect to protein amount Broad linear range (above 3 orders of magnitude) detect low and high abundance proteins in the same exposure Long lasting signal carry out exposures at your convenience Versatility optimized for CCD imaging and compatible with film detection Flexible high sensitivity with chemiluminescent or chemifluorescent imaging with laser based fluorescence imaging systems (e.g. Typhoon imager) Product name Size/description Product code Use Optiblot buffers with Optiblot gels for optimal results ECL Detect Kit (23pg-187ng) 20ml, 200ml or 500ml ab ECL Max Detect Kit (4.6pg-4.7ng) 20ml, 100ml, or 200ml ab ECL Ultra Detect Kit (1.2pg-2ng) 20ml, 100ml, or 200ml ab

5 Fluorescent western blotting: Substrates for fluorescent western blotting: Through advanced chemistry Optiblot ECL Detect kits provide imaging flexibility, with emitted light that can be detected using fluorescent imaging systems in addition to film and CCD based systems. Product name Size/description Product code ECL Max Detect Kit (4.6pg-4.7ng) 20ml, 100ml, or 200ml ab ECL Ultra Detect Kit (1.2pg-2ng) 20ml, 100ml, or 200ml ab Optiblot electrophoresis kits: Includes 10x gels and all buffers required for rapid and economical protein electrophoresis Available for studying denatured proteins (Reducing Kits) or native proteins (Non- Reducing Kits) Gels a vailable in 12 well format and compatible with commonly available tanks Optiblot Reducing Electrophoresis kits: Product name (gel size and concentration) Product code Optiblot Fluorescent Western Blot Kit (ab133410): All reagents you need for fluorescent detection of two proteins on the same blot at the same time. High sensitivity detect low abundance proteins Timesaving protocol (3.5 hours) - no need for stripping and re-probing Detect phosphorylated and non-phosphorylated isoforms simultaneously Sufficient for 10 assays Compatible with CCD imagers equiped with Cy3/Cy5 excitation and emission: ChemiDoc MP, LAS 4000, Fluorchem Q and M systems, G:BOX, Gel Logic, Fusion FX5, Typhoon imager and similar systems. Fluorescent detection gives you increased throughput Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (10%) Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (12%) Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (4-8%) Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (4-20%) Optiblot Reducing Electrophoresis Kit - 8 x 10 cm (10%) Optiblot Reducing Electrophoresis Kit - 8 x 10 cm (12%) Optiblot Reducing Electrophoresis Kit - 8 x 10 cm (4-8%) Optiblot Reducing Electrophoresis Kit - 8 x 10 cm (4-20%) Optiblot Non-reducing Electrophoresis kits: Product name (gel size and concentration) Optiblot Non-reducing Electrophoresis Kit - 10 x 10 cm (10%) Optiblot Non-reducing Electrophoresis Kit - 10 x 10 cm (12%) Optiblot Non-reducing Electrophoresis Kit - 10 x 10 cm (4-8%) Optiblot Non-reducing Electrophoresis Kit - 10 x 10 cm (4-20%) Optiblot Non-reducing Electrophoresis Kit - 8 x 10 cm (10%) Optiblot Non-reducing Electrophoresis Kit - 8 x 10 cm (12%) Optiblot Non-reducing Electrophoresis Kit - 8 x 10 cm (4-8%) Optiblot Non-reducing Electrophoresis Kit - 8 x 10 cm (4-20%) For a complete product listing visit: ab ab ab ab ab ab ab ab Product code ab ab ab ab ab ab ab ab

6 Optiblot FAQs Optiblot Gels and Buffers 1. Are Optiblot Run Buffers Tris-MOPS or Tris-Tricine buffers? Optiblot Reducing SDS Run Buffer (ab119195) is a reducing Tris-MOPS buffer system similar to conventional MES buffers (excellent for separation of low molecular weight proteins). Optiblot SDS Run Buffer (ab119197) is a non-reducing Tris-tricine buffer system providing similar separation to MOPS buffers (increased separation of high molecular weight proteins). 2. What are the recommended running conditions for Optiblot gels and buffers? Voltage Start current Ending current Run time 180V 90mA/gel 40mA/gel minutes * For compatible tanks 3. Do I need to use Optiblot buffers? Yes, Optiblot buffers must be used with Optiblot gels to achieve the best results. Other buffer systems will result in poor electrophoresis. Buffer recipes are available online. 4. How many gels can be run with a 500ml bottle of Run Buffer? Optiblot Run Buffer is 20x concentrated and 500ml is sufficient for a full box of gels (i.e. 40ml Run Buffer per gel). Optiblot Blue 1. Do I need to do anything prior to staining my gel? No, simply remove the gel from the cassette and place directly in the staining solution. 2. How long does Optiblot Blue take? You can expect to see the strongest bands appearing after around 5 minutes with all bands being visible after 15 minutes. Total saturation of Optiblot Blue occurs after approximately 1 hour. 3. Do I still need to de-stain my gel? No, Optiblot Blue has been formulated not to bind to the SDS or Acrylamide in the gel, providing a clear background. 6. What is the sensitivity of Optiblot Blue? Optiblot Blue can clearly visualize a 5ng band of BSA. However, this may not be the case for your protein and is also dependent on basicity - the more basic your protein the better the sensitivity. Typically, staining is in the range of 5 to 25 ng protein per band. 7. Is Optiblot Blue compatible with mass spectrometry (MS) and Edman sequencing? Yes. Optiblot Bradford Reagent 1. Can I use Optiblot Bradford Reagent if my sample contains detergents? Yes, Optiblot Bradford Reagent can resist the effects of detergents which normally influence standard Bradford assays. 2. Which standards should I use for my standard curve? The Bradford assay is affected by the basicity and hydrophobicity of the protein. Typically, proteins have a basic amino acid composition of 10 to 17 mol% and you should try to use a standard as close to the mol% of basic amino acids as your unknown protein (you may need to determine the hydrophobicity of your protein). Some good standards are shown below. Standard Protein Mol% positive residues Hen egg white lysozyme 13.9 Bovine serum albumin (BSA) 16.5 Bovine Immunoglobulin (IgG) 11.3 Bovine beta lactoglobulin 11.8 Contact Abcam scientific support for more information 4. How do I dispose of Optiblot Blue? Optiblot Blue is non-toxic and can typically be disposed of in the sink. However, always check your local regulations. 5. Will my gels over-stain? No, Optiblot Blue will not cause gels to be over-stained, even if left submerged for days. 9 10

7 Western blot troubleshooting tips Contents 1. No signal 2. High background 3. Multiple bands 4. Uneven white spots on the blot 5. Black dots on the blot 6. White bands on a black blot (negative of expected blot) 7. MW marker lane is black 8. The band of interest is very low/high on the blot 9. Smile effect of the bands 10. Uneven band size in lanes probed for the same protein 11. Uneven staining of the blot 12. Heavy chain is obstructing my band of interest 1. No signal 1.1. The primary antibody and the secondary antibody are not compatible. Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary) Not enough primary or secondary antibody is bound to the protein of interest. Use more concentrated antibody, or check datasheet suggestions. Incubate longer (e.g. overnight) at 4ºC Cross-reaction between blocking agent and primary or secondary antibody. Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents are milk, BSA, serum or gelatin) The primary antibody does not recognize the protein in the species being tested. Check the datasheet or perform a ClustalW alignment to ensure your antibody should react with the target protein; Run the recommended positive control Insufficient antigen. Load at least μg protein per lane (check protein quantity using BCA or Bradford Reagent (ab119216); Use protease inhibitors (ab65621); Run the recommended positive control The protein of interest is not abundantly present in the tissue. Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.). Check that the protein is expressed in the tissue, or include a lane for a positive control if a sample known to express the protein of interest is available. Or try using a more sensitive ECL such as ECL Max Detect (ab133408) or ECL Ultra Detect (ab133409) Poor transfer of protein to membrane. Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the wrong way; if using PVDF membrane make sure you pre-soak the membrane in MeOH then in transfer buffer. Run the transfer for longer Excessive washing of the membrane. Do not over wash the membrane. Too much blocking does not allow you to visualize your protein of interest. Instead of using 5% milk in the buffer try removing the milk or using 0.5%. Switch blocking reagents or block for less time Over-use of the primary antibody. Use fresh antibody as the effective concentration is lowered upon each re-use Secondary antibody inhibited by sodium azide. Do not use sodium azide in the dilution buffer with HRP-conjugated antibodies. A detailed guide to western blot Detection kit is old and substrate is inactive. Use fresh substrate Inefficient transfer Check for transfer efficiency by incubating membrane in Ponceau S. This also allows you to check for air bubbles, unequal loading and overloading of the gel. 2. High background 2.1. Blocking of non-specific binding might be absent or insufficient. Increase the blocking incubation period and consider changing blocking agent. Abcam recommends 5% nonfat dry milk, 3% BSA, or normal serum for 30 min. These can be included in the antibody buffers as well The primary antibody concentration may be too high. Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best). Check datasheet for suggestions Incubation temperature may be too high. Incubate blot at 4 C The secondary antibody may be binding non-specifically or reacting with the blocking reagent. Run a secondary control without primary antibody Cross-reaction between blocking agent or buffer and primary or secondary. Add a mild detergent such as Tween 20 (Product code: ab64028) and phosphatase inhibitors to the incubation and washing buffer (for phospho-specific proteins). Milk contains casein which is a phosphoprotein; this is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Use BSA as a blocking reagent instead of milk. In addition for phospho-specific proteins do not use phosphate containing buffers Washing of unbound antibodies may be insufficient. Increase the number of washes Your choice of membrane may give high background. Nitrocellulose membrane is considered to give less background than PVDF The membrane has dried out. Care should be taken to prevent the membrane from drying out during incubation. 3. Multiple bands 3.1. Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles. Go back to the original non-passaged cell line and run the current and original cell line samples in parallel The protein sample has multiple modified forms in vivo such as acetylation, methylation, myristylation, phosphorylation, glycosylation etc. Examine the literature and use an agent to dephosphorylate, de-glycosylate, etc. the protein to bring it to the correct size The target in your protein sample has been digested (more likely if the bands are of lower molecular weight). Make sure that you incorporate sufficient protease inhibitors in your sample buffer Unreported novel proteins or different splice variants that share similar epitopes and could possibly be from the same protein family are being detected. Check the literature for other reports and also perform a BLAST search; Use the cell line or tissue reported on the datasheet. Use the appropriate control to check expression of the protein ( Primary antibody concentration is too high - at high concentration multiple bands are often seen. Try decreasing the antibody concentration and/or the incubation period

8 3.6. Secondary antibody concentration is too high - at high concentration secondaries will bind nonspecifically. Try decreasing the concentration. Run a secondary antibody control (without the primary) The antibody has not been purified. Try to use affinity purified antibody. This will often remove non-specific bands The bands may be non-specific. Where possible use blocking peptides to differentiate between specific and non-specific bands. Only specific bands should be blocked (and thus disappear) The protein target may form multimers. Try boiling in SDS-PAGE for 10 minutes rather than 5 minutes to disrupt multimers. 4. Uneven white spots on the blot 4.1. Air bubbles were trapped against the membrane during transfer or the antibody is not evenly spread on the membrane. Make sure you remove bubbles when preparing the gel for transfer. Incubate antibodies under agitation. 5. Black dots on the blot 5.1. The antibodies are binding to the blocking agent. Filter the blocking agent. 6. White bands on a black blot (negative of expected blot) 6.1. Too much primary and/or too much secondary antibody. Dilute the antibodies more. 7. MW marker lane is black 7.1. The antibody is reacting with the MW marker. Add a blank lane between the MW marker and the first sample lane. 8. The band of interest is very low/high on the blot 8.1. Separation is not efficient. Change the gel percentage: a higher percentage for small protein, lower percentage for large proteins Post translational modifications such as phosphorylation or ubiquitylation lead to an increase in the MW of the protein of interest. In the case of ubiquitylation a distinct smear can be observed. 9. Smile effect of the bands 9.1. Migration was too fast or migration was too hot (changing the ph and altering the migration). Slow down the migration or run the gel in the cold room or on ice. 10. Uneven band size in lanes probed for the same protein 10.1.Gel has set too quickly while casting and the acrylamide percentage is not even along the lanes. Review the recipe of the gel and the addition of TEMED to the gels, add a little 0.1% SDS in water to the top of the migrating gel while it sets to stop it from drying. 11. Uneven staining of the blot Contamination from bacteria Keep antibodies at 4 C and use fresh buffers covers the gel Not enough antibody Make sure the membrane is covered with the antibody/ incubate under agitation. 12. Heavy chain is obstructing my band of interest 12.1.When performing a western blot of IP samples the secondary antibody is recognizing both Heavy (~50 kda) and Light chains (~25 kda). Use a light chain specific secondary antibody that doesn t recognise the heavy chain. ( All your western blot needs covered Sample preparation Electrophoresis Membrane transfer Antibody incubation Detection Primary antibodies tested in western blot SDS-PAGE gels Buffers and accessories Electrophoresis kits Cell fractionation kits Blocking reagents Western blot antibody cocktails ECL substrate kits EasyLink antibody conjugation kits Transfer buffers Fluorescent detection reagents PVDF membranes Nitrocellulose membranes AbExcel secondary antibodies Protein ladders Lysates and loading controls Bradford reagent BCA kits Buffers SDS-PAGE clean up kits VeriBlot for IP secondary antibodies Key: = = = = = = = =

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