Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules. All blotting techniques share a similar workflow. Initially, an electrophoretic procedure is used to separate molecules (or protein and nucleic acid fragments) by size on a gel based on the movement of macromolecules in an electric field. Then, these separated molecules are transferred to a solid membrane (nitrocellulose, nylon, polyvinylidene difluoride (PVDF), etc.) suited to immobilize the target molecule of interest. End-users are then able to incorporate “labels” (radiolabel, fluorescent label, reporter enzyme label) to the immobilized targets with probes that are sequence-specific or shape-specific to the target molecule of interest (either DNA, RNA or protein) during an incubation step that facilitates hybridization. This hybridized complex (“immobilized target molecule--label/probe”) which consists of a bound probe can be later detected and visualized using various imaging methods. Below, we review some considerations between Southern, Northern and Western blots used to detect DNA, RNA or protein, respectively.
Detection of Nucleic Acids by Southern and Northern Blotting
Southern blotting was originally introduced by Edwin Southern in 1975. It is an analytical technique in molecular biology research that end-users use to measure the size and amount of specific DNA sequences in a complex mixture through immobilization of the target sequence to a solid-support followed by hybridization of a complementary DNA probe. Shortly after southern blotting was developed, the premise of this analytical technique was applied to the measurement of the size and amount of RNA transcripts from a gene of interest which is known as Northern Blotting. Both blotting methods share a similar workflow 1) Sample preparation of purified high quality DNA or RNA from a sample, 2) Gel electrophoresis to separate nucleic acid fragments by size, 3) transfer (“blotting”) to a solid-support that immobilizes isolated target nucleic acid, 4) preparation & hybridization of nucleic acid probe, and lastly 5) the detection of the nucleic acid probe.
During sample preparation, Southern and Northern blots both begin very similarly - the target DNA or RNA is purified from samples through nucleic acid extraction. Purified whole genomic DNA, plasmid DNA or RNA transcripts are isolated using common extraction techniques such as organic extractions (phenol-chloroform and ethanol precipitation), filter-based, spin basket extractions (glass fiber, derivitized silica or ion exchange column-based method) and magnetic particle methods.
After extraction, Southern and Northern blot protocols begin to differ. Southern blotting requires an additional step because of the need to take purified DNA and partially digest it into smaller, different sized, double-stranded fragments with restriction enzymes (endonucleases) that cut the DNA at specific spots. DNA samples are digested with appropriate restriction enzyme for 2-24h at 37°C. If DNA is clonally derived, a digestion time of 1-2h may be sufficient. Northern blots omit the need for restriction digest. However, RNA detection by Northern blot requires different considerations and a pretreatment step because the end-user is using RNA which is naturally unstable. Given the instability of RNA, procedures need to be done with sterile, RNAse-free supplies that usually require reagents to be autoclaved or filter-sterilized; for autoclavable plastics – 20 mins at 120°C to inactivate RNAses, for non-autoclavable plastics – 2 or more hours in 0.1% diethyl pyrocarbonate, glassware needs to be heated at 180°C for 3 h. After extraction for Northern blotting, already single-stranded RNA needs to be pretreated with formaldehyde or a denaturing solution with glyoxal to prevent the formation of base-paired secondary structures. Glyoxal reacts with guanine and introduces an additional ring that prevents base-pairing by steric hindrance. While formaldehyde reacts with free amines of the bases producing a Schiff base that is unable to hydrogen bond to complementary baes preventing the formation of secondary structures. The amount of target RNA needed to be yielded from extraction depends on how present that specific mRNA species is within the mixture of cellular RNA species present. For low abundance mRNA, 1-10μg of poly (A) enriched RNA is used and for medium to high abundance 10-20μg of total RNA is sufficient to detect a signal.
Once samples are prepared, both protocols take the different sized, single-stranded, target nucleic acid fragments and load them into wells with appropriate DNA or RNA ladders for gel electrophoresis to be separated by size. The fragments are loaded into wells in an agarose or polyacrylamide gel. For higher resolution of shorter fragments (<100bp) polyacrylamide gels are frequently used over agarose gels . As the electric current is applied to the gel matrix, DNA or RNA which has a negatively charged backbone, will migrate from the cathode towards the anode. Smaller fragments migrate faster and farther on the gel than larger fragments. Generally, agarose concentrations of 0.5% - 2% are used for separation of fragments of 100 base pairs (bp) up to several kilobases (kb). For better separation of larger molecules (more than 750kb), agarose concentration has to be lowered to 0.5%. For better separation of smaller fragments (less than 500bp) – 1.5 to 2%. Typically, a voltage of 100mV (5 -8 V/cm) is applied to a gel. Applying higher voltages may reduce the run time for a gel but it may also come at the cost of a decrease in resolution. Common buffers used for agarose electrophoresis are in tris-acetate EDTA (TAE) and sodium boride (SB). TAE has a low buffering capacity but provides the best resolution. SB has the highest buffering capacity, allowing shorter run times for fragments smaller than 5kb (up to 350mV). After the fragments have been separated by electrophoresis, they are visualized by adding ethidium bromide to the gel and running the gel in a UV transilluminator.
Then, these single-stranded fragments are transferred from gel onto a nitrocellulose or nylon membrane. End-users can choose to transfer by placing the membrane on top of the gel that lies on a stack of wet filter paper and applying pressure (either suction or weight on top of the membrane and gel) physically or within an electrophoretic chamber. If using a nitrocellulose membrane, the membrane is baked in a vacuum oven. If using a nylon membrane, it is exposed to ultraviolet light to encourage DNA or RNA to covalently cross-link the solid support. Nylon-based membranes are preferred over nitrocellulose for northern and southern blotting because nylon has a higher binding affinity to nucleic acids and is provides greater physical strength as a solid support. Charged nylon membranes offer the highest nucleic acid binding affinity for northern and southern blotting.
Following transfer, the membrane is prepared for hybridization of the nucleic acid probe by being washed with a high concentration of DNA (herring or salmon sperm DNA) which prevent nonspecific interactions between the membrane’s material and nucleic acids. After blocking, the membrane is incubated with a single-stranded nucleic acid probe that has a complementary sequence to the target DNA or RNA and is labeled with a radioisotope, fluorescent molecule or enzymatic reporter. Common probes for Northern and Southern are radioactive isotopes such as 32
P or 125
I, fluorescent or digoxigenin (DIG)-labeled DNA or RNA reverse complement. During the incubation, this probe will hybridize to the target immobilized DNA or RNA forming probe-fragment complexes that remain completely fixed to the blot. The hybridized probes are then visualized through activation of the label apart of the probe-fragment which generates a detectable chemiluminescent signal that can be captured and measured.
Detection of Proteins by Western Blot
Western blotting is a lab technique that characterizes the amount, size and identify of specific proteins within a sample matrix. Western blot protocols share the similar steps of other blotting protocols; preparation of sample lysates to isolate proteins, gel electrophoresis to separate proteins by size, transfer to a membrane followed by hybridization of an antibody-based probe.
Sample preparation for the assay begins with lysing samples in a protein extraction buffer with proteinase inhibitors and homogenizing samples then removing cellular debris via centrifugation. The resultant lysates are then assayed for whole protein content via protein quantification assay (ex. Bradford assay). Before moving on to electrophoretic procedures, samples have to be boiled and treated with a buffering solution that contains Sodium dodecyl sulfate (SDS) and reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol. The heat and disulfide reducing agents break disulfide bonds which help to maintain protein tertiary and quaternary structures. SDS is an anionic detergent that further denatures the proteins isolated by deteriorating secondary and non-disulfide tertiary structures to completely unfold the protein into a linearized, denatured product for electrophoresis.
Denatured protein sample lysates are then placed into wells for polyacrylamide gel electrophoresis and measured for size using a molecular weight standard in a separate lane. A 5-50mA current is applied across the gel and proteins are separated through movement in the resolving gel which produces bands. Resolving gels are made in various acrylamide concentrations depending on the target size of the protein.
After electrophoresis, a transfer unit/cassette is assembled that includes the gel with separated proteins and transfer membrane (nitrocellulose or PVDF) sandwiched between filter paper and padding, assembled cathode to anode for electrotransfer in a transfer buffer. Proteins are transferred from gel to membrane within the transfer apparatus towards the anode (positive electrode) at 200-300mA for 2h to overnight. Higher molecular weight proteins and high acrylamide concentrations require longer transfer times. Transfer efficiency can be checked via stain in Ponceau S solution before preparing the membrane for detection of proteins.
Following transfer, the membrane is prepared for antibody staining by incubation with a blocking solution which prevents nonspecific binding. Typically 5% nonfat dry milk or 3% bovine serum albumin (BSA) are used for blocking and are in solution with a detergent. This is then followed by incubation of the membrane with primary antibody to target of interest. This facilitates the direct detection of the protein of interest.. Utilizing a secondary antibody that targets the primary antibody Fc region (species specific) and is linked to an enzyme that catalyzes a chemluminescent light signal can provide the signal amplification to visualize very small quantities of protein. Secondary antibodies are conjugated to biotin or enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). When the enzyme substrate is added, either a colored precipitate (colorimetric detection) or a chemiluminescent or fluorescent product is formed and the light signal is captured.
We offer multiple Western Blot detection kits (Anti-mouse & anti-rabbit
: ENZ-KIT163, Anti-mouse
: ENZ-KIT182, Anti-rabbit
: ENZ-KIT183) to save you time, lower your costs, and eliminate waste. Get your results at the bench without needing a developer or high-end imaging instruments. The kit comes with secondary antibodies, antibody blocker/diluents and wash buffers compatible with anti-mouse and anti-rabbit primary antibodies (see kit details for species reactivity). These kits can detect low expressing proteins with high sensitivity (Figures 1, 2, and 3). We offer additional accessories as well such as a pre-stained protein ladder
(ENZ-ACC131) and thousands of primary antibodies
|Target for Detection
||DNA extraction, enzymatic digestion by restriction enzymes
||RNA isolation, denaturation w. formaldehyde
||Protein extraction, protein denatured with SDS
||Agarose Gel Electrophoresis
||Agarose Gel Electrophoresis
||Nitrocellulose or PVDF
||Nucleic acid probe w/ single stranded sequence homologous to target DNA
||RNA, DNA or oligodeoxynucleotide
||Primary antibody(Direct), Primary + Secondary (Indirect) conjugated to fluorophore, reporter enzyme
||X-ray film, Chemiluminescence
||X-ray film, Chemiluminescence
||CCD Camera, LED or Infrared imaging
Comparisons between Northern, Southern and Western Blots.
Figure 1. Visual Reading for Rapid Results using WESTERNVIEW? Detection kits. Total cell lysate from Jurkat cells. Exposure to NBT/BCIP for 1 minute.
Figure 2. Detect Difficult Proteins. Jurkat cells treated with 12.5 μM etoposide for 18 hours. Loaded 12 μg lysate per well. Using WESTERNVIEW? Detection kits.
Figure 3. PANC-1 cells treated with etoposide. The band at 50 kDa is clearly revealed to be tubulin, while the smaller bands are known to be cleaved caspase.
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