This protocol has been optimized using commercially produced buffers, gels and transfer stacks in order to reduce variability and improve consistency. Refer to Materials List for a complete list of consumables required.
Fluorescent WB protocol using I-Blot fast transfer and LI-COR Odyssey imaging system
1. Preparation of Sample
Figure 1. Positive control selection. The addition of positive controls to an experiment confirms the labeling detected is real. However, caution must be taken to ensure your control is working correctly prior to using experimental samples. A) Fusion protein TREM 2 was loaded at the manufacturer’s guidelines of 1 μg/ml, however the labeling observed at 110 kDa conflicts with the datasheet predicted molecular weight of 60-70 kDa. B) After addition and incubation with the reducing agent, the fusion protein labeling was detected at the predicted molecular weight. However, this meant a greater protein load was required as the reduction process decreased the signal of the protein.
2. Electrophoretic Separation of Proteins
3. Total Protein Stain of the Loading Control Gel
4. I-Blot Semi Dry Fast Protein Transfer
NOTE: All reagents required for protein transfer using the I-Blot machine are commercial products specifically designed for the I-Blot methodology and can be found in the Materials List.
Figure 2. Optimization of transfer using the I-Blot. A) A single gel loaded with ladder and 15 μg of murine whole brain homogenate in three tandem repeats was cut into three sections. One section was not transferred, one was transferred for 7.5 min (as per manufacturer's guidelines) and one for 8.5 min. The gel sections were then stained with Instant Blue protein stain, scanned on an infrared imager in the 680 channel and quantified. B) Graphical representation of the quantification values demonstrating the difference in residual protein content of each gel following 0, 7.5, and 8.5 min of transfer. Note that an additional minute of transfer time resulted in additional protein transfer of approximately 45%. Please click here to view a larger version of this figure.
5. Antibody Detection of Proteins
Figure 3. Optimization of secondary antibodies. A) A multi species comparison of secondary only non-specific labeling against 15 μg of murine (M), Ovine (O) and Equine (E) nervous tissue homogenates with a variety of fluorescent tagged secondary antibodies. L is the molecular weight ladder. Ovine tissue homogenate was the only sample to cross-react with the secondary labeling when using donkey anti-goat 800 antibody. B) It is also important to ascertain if non-specific labeling occurs when using a different tissue sample, i.e., murine gastrocnemius muscle (15 μg load). This sample cross reacts with the donkey anti-mouse 680 secondary antibody, however this did not occur when using mouse brain homogenate.
Figure 4. Troubleshooting secondary antibody specificity. Western blot of a range of tissue samples (15 μg protein per lane) — fat, muscle, liver and bone — incubated with ERK primary antibody and incubated with three different secondary antibodies. Top panel: WB labeled with LI-COR goat anti-rabbit 680 secondary antibody produced weak labeling of fat and bone and no signal was detected in the liver and muscle samples. Middle panel: Membrane (from top panel) was stripped and reprobed using ECL methodology and Goat anti-rabbit HRP linked secondary. Bands are now visible in muscle and liver samples and labeling appears more intense in the fat and bone samples. Bottom panel: Membrane (from top and middle panel) was stripped and reprobed using LI-COR Donkey anti-rabbit 680 secondary antibody which has shown a greater affinity for the ERK primary antibody. Labeling for muscle and liver is now visible with increased signal intensity from both fat and bone samples. Please click here to view a larger version of this figure.
6. Visualization
NOTE: All images are acquired using the LI-COR Odyssey Classic imager and associated Image Pro analysis software (version 3.1.4).
Figure 5. Visualization and quantification of western blot. Scan of a western blot showing murine gastrocnemius muscle (30 μg load) probed with Annexin V primary antibody (36 kDa) and goat anti-rabbit 800 secondary antibody. The membrane was scanned and visualized in the 800 channel. To quantify the protein (Annexin V), a rectangular box is drawn around the band of interest from sample 1. This is then copied and pasted over the remaining sample lanes to ensure measurement of the same area. Background is automatically accounted for around the shape drawn but this can be altered to ensure the background measurement is accurately defined. The table below displays the quantified measurements of each shape drawn including total signal obtained, background and signal with background subtracted. This information can then be exported into a spreadsheet program to calculate expression ratios (as determined by relative fluorescence intensity) and allows subsequent statistical analyses to be performed. Please click here to view a larger version of this figure.
7. Post Visualization
As QFWB sensitivity and the linear range of detection is greater than conventional ECL detection, there are a number of control measures that are crucial to ensure that accurate data is collected, thereby aiding effective interpretation. Firstly, the inclusion of positive control samples as shown in Figure 1. Secondly, optimization of transfer to guarantee equivalent movement of high and low molecular weight proteins from the gel to the membrane as exhibited in Figure 2. Thirdly, optimization of antibodies, especially secondary antibodies whose optimization is often overlooked, but which can produce non-specific banding capable of interfering with correct interpretation of protein(s) of interest. See Figure 3. Fourthly, it may also be the case that when a protein appears undetectable but is expected to be present, this may also be a secondary antibody issue which can be corrected by simply using a secondary raised in a different species host. See Figure 4. Fifthly, total protein labeling and analysis is a far more robust and quantifiable method in comparison to the use of traditional single protein(s) that are ubiquitously expressed for internal reference standards3. Many of these single proteins have been found to be differentially expressed in models of neurodegenerative diseases as well as between different tissue samples and the uniformity of expression can alter within the same tissue3. Therefore, production of a loading control gel will confirm the uniformity of sample load when combined with a total protein analysis by comparing and quantifying the protein load in each lane at various molecular weights ranges measured against each sample to indicate standard error as demonstrated in Figure 6. Importantly, all of these troubleshooting techniques and controls are only as effective as the sensitivity and consistency of the analysis tools applied by the operator (Figure 5). Finally this technique lends itself to stripping and re-probing of membranes with more flexibility than ECL due to factors including but not limited to increased sensitivity, reduced background, dual color detection and membrane stability under long term storage conditions. See Figure 7.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
RIPA Buffer | Fisher Scientific UK Ltd | 10230544 | |
M Tubes | Miltenyi Biotec Inc. | 130-093-236 | |
iBlot Transfer Stack, PVDF Regular | Life technologies, UK | IB401001 | |
MagicMark XP Western Protein Standard (20-220 kDa) | Life technologies, UK | LC5602 | Use in gel 1 for Western blotting |
SeeBlue Pre-stained protein standard | Life technologies, UK | LC5625 | Use in gel 2 Total protein stained gel |
NuPAge LDS Sample buffer 4X | Life technologies, UK | NP0007 | |
NuPAGE MES SDS Running Buffer (for Bis-Tris Gels only) (20X) | Life technologies, UK | NP0002 | |
NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well | Life technologies, UK | NP0322BOX | |
PHOSPHATE BUFFERED SALINE TABLET,*TRU-ME, PHOSPHATE BUFFERED SALINE TABLET | Sigma-Aldrich, UK | P4417-100TAB | |
Micro BCA, Protein Assay Kit | Fisher Scientific UK Ltd | 10249133 | |
Odyssey blocking buffer | Li-Cor Biosciences | P/N 927-40000 | |
IRDye 680RD Goat anti-Rabbit IgG (H+L), 0.5 mg | Li-Cor Biosciences | 926-68071 | |
IRDye 680RD Donkey anti-Mouse IgG (H+L), 0.5 mg | Li-Cor Biosciences | 926-68072 | |
IRDye 800CW Goat anti-Rabbit IgG (H + L), 0.5mg | Li-Cor Biosciences | 926-32211 | |
IRDye 800CW Donkey anti-Goat IgG (H + L), 0.5mg | Li-Cor Biosciences | 926-32214 | |
ODYSSEY CL Infra-red imager | Li-Cor Biosciences | Call for quotation | |
iBlot 7-Minute Blotting System | Life technologies, UK | This model is no longer in production | |
InstantBlue Protein stain | Expedeon, UK | ISB1L | |
Revitablot western blot stripping buffer | Rockland Immunochemicals Inc. | MB-085-0050 |
The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term “western blot” suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many “optimized” western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.
The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term “western blot” suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many “optimized” western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.
The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term “western blot” suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many “optimized” western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.