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This is the general process of the Western blotting experiment. We will introduce each step in detail in the following text.
Fig. 1. Flowchart of Western blot[1].
1.Protein sample preparation
1.1 Protein extraction
(1) Prepare solution: melt protein lysate at room temperature RIPA , add Protease Phosphatase Inhibitor mixture (50X) to give a final concentration of 1X, mix well and place on ice immediately.
(2) For adherent cells: wash 2 to 3 times with pre-cooled PBS at 4°C, scrape off the cells with a cell scraper or treat the cells with EDTA solution so that the cells are no longer adhering very tightly to the wall, and blow down the cells with a pipette. Collect the cells by centrifugation and aspirate the supernatant to the best of your ability, leaving the cell precipitate for backup. For suspended cells: wash 2~3 times with PBS, collect cells by centrifugation, aspirate the supernatant to the best of your ability, and leave the cellular precipitate for backup. For tissue samples: cut an appropriate amount of tissue and an appropriate amount of mixed protein lysate in a homogenizer and grind well (0.01g of tissue plus 50-100ul of protein lysate) until no tissue clumps are visible.
(3) Lysis: Cell samples were lysed at 1x106 cell number with 100μL of lysis solution, and lysed on ice for 30min (or lysed on ice for 5min and sonicated on ice bath for 20s). Tissue samples were transferred to a 1.5 ml EP tube and lysed on ice for 15 min.
(4) Extraction: centrifuge at 12,000 rpm and 4℃ for 10 min. Immediately transfer the supernatant to a pre-cooled 1.5 ml EP tube to extract cell plasma proteins.
Tips of Protein Extraction
1. Tissue or cell selection and processing: Make sure that the selected tissue or cell samples are fresh and representative. For tissue samples, unwanted parts, such as fat and blood vessels, need to be removed. For cell samples, it is needed to ensure that the cells are in good condition and free of contamination.
2. Selection and use of lysate: The selection of lysate should be based on the type of sample, the purpose of the experiment and the nature of the desired protein. When lysing cells/tissues, it is necessary to add protease inhibitors to the lysate in advance to inhibit the degradation of cellular proteins by endogenous proteases. When detecting phosphorylation, it is necessary to add additional phosphatase inhibitors, and it is best to add them as they are used. Commonly used RIPA buffer (strong) formulations: 1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA.
3. Low-temperature operation: The entire protein extraction process should be carried out at low temperatures to prevent protein degradation. An ice box, ice bath or refrigerated equipment can be used to maintain a low temperature environment.
4. Sample preservation: avoid repeated freezing and thawing of proteins obtained by extraction. For long-term storage they should be divided and placed in a -80°C refrigerator[2].
1.2 BCA protein concentration measurement
(1) Prepare the BSA standard and prepare BCA working solution A: B = 50:1. The samples were diluted with pre-cooled PBS.
(2) The standards and samples were spiked into a 96-well plate.
(3) Add BCA working solution to each well and incubate at 37 ℃ for 30 min.
(4) The OD value of each well was read at 562 nm on the zymograph.
(5) The protein concentration of the sample was calculated from the standard curve and the sample volume used.
2. Electrophoresis
2.1 Gel preparation
(1) Prepare the glass plate, ensure that the lower edge of the two glass panels are horizontal and no openings, the installation of the inner length of the outer short, compacted by hand the glass plate and vertical groove of the adhesive frame.
(2) Tilt the vertical slot glue-making rack into the vertical slot glue-making fixed frame, pay attention to prevent the film deformation leakage of liquid, do not overly force downward pressure, and ensure that the card is good.
(3) Prepare separation gel (1 mm thickness) in accordance with 5 ml per block, mix gently and spread the plate immediately.
(4) Use isopropyl alcohol to flatten the liquid surface of the separation glue after spreading the board.
(5) After 40 min of solidification, aspirate the isopropanol. Rinse gently with ddH2O and blot dry.
(6) Dispense the gel concentrate according to 1.5 ml per block and insert a comb of the appropriate size, taking care that the operation is rapid and horizontal and that no air bubbles are generated.
(7) After the 40min concentrated gel is solidified, remove it from the gel-making rack, clean the residual gel on the gel plate with ddH2O, and wrap it in plastic wrap for immediate use or 4°C storage.
2.2 Sample preparation
Add 4xsampling buffer and mix well, leave it at 100℃ for 10 min, then cool it quickly in ice bath.
2.3 Performing gel electrophoresis
According to the results of BCA, the sample volume of the target protein was 20-40μg per well, and the sample volume of the internal reference protein was 5-10μg per well. low voltage electrophoresis was used for the upper layer of the gel, and the voltage was adjusted to 80V by turning on the power supply, and high voltage electrophoresis was used when the bromophenol blue entered the lower layer of the gel, and the electrophoresis was stopped when the voltage was adjusted to 120V until the bromophenol blue reached near the bottom of the gel.
Tips of Electrophoresis
1. Cleaning of the glass plate: The glass plate must be thoroughly cleaned to avoid any residue affecting the electrophoresis results. It is recommended to use deionized water for rinsing and ensure air drying or baking. This step is critical as it ensures uniformity and consistency in the electrophoresis process.
2. Accuracy of glue dispensing: accurately dispense separated glue and concentrated glue according to the instructions. Be careful to shake and mix gently when dispensing to ensure the quality and stability of the glue. In addition, AP should be prepared and used now to avoid deterioration and non-polymerization of the glue.
3. Cleaning of Sampling Wells: Before adding samples, be sure to use a pipette gun to blow wash each sampling well to remove residual acrylamide residue. This will avoid the appearance of aberrant bands and ensure the accuracy of the electrophoresis results.
4. Temperature control: The temperature of the gel during electrophoresis should be properly controlled. Excessive temperatures may cause the bands to develop a "smile" shape, i.e., the sides of the bands curve upward. Therefore, if necessary, the electrophoresis bath can be kept at a low temperature in an ice box.
5. Selection of voltage: The smaller voltage will make the molecular sieve effect of the gel fully realized. Conventional electrophoresis conditions are concentrated gel 80V, 30min, separated gel 120V constant voltage until strip differentiation. In order to reduce the chance of band bending, 80V constant voltage can be selected, and the uniformity of the band will be greatly improved, and the disadvantage is that the experiment time will be extended.
3. Membrane Transfer
(1) The transfer buffer was pre-cooled to 4°C in advance.
(2) The PVDF membrane should be activated with methanol for 30s before use, and then placed in the membrane transfer buffer.
(3) Place the filter paper and gel together in the membrane transfer buffer.
(4) Place the sponge, filter paper, gel, PVDF membrane, filter paper and sponge in order according to the sandwich method and carefully remove all air bubbles.
(5) The PVDF membrane is located on the anode side and the gel is located on the cathode side, inserted into the electrophoresis tank and poured into the membrane transfer buffer.
(6) Set the transmembrane current to constant current, 220mA, and set the transmembrane time according to different protein sizes.
Tips of the Membrane Transfer
1. Selection of membrane: The commonly used transfer film mainly includes PVDF film and NC film.
PVDF membrane can provide better protein retention rate, physical strength and wide chemical compatibility, it is more recommended; PVDF membrane has two specifications, which can be selected according to the molecular weight size of the target protein.
NC membrane has a very low background and is relatively inexpensive. The disadvantages are that for small molecular weight proteins will be lost during washing, and the NC membrane is less tough, easy to break during operation, and requires more skill in operation.
2. Pre-treatment of membranes: Membranes need to be properly pre-treated. For the PVDF membrane, it is completely soaked in methanol for 30 seconds to activate the positively charged groups on the membrane, promoting binding to the negatively charged proteins. NC membranes, on the other hand, need to be equilibrated in transfer buffer for some time. In addition, in order to easily distinguish the positive and negative of the membrane, the upper right corner of the membrane can be processed by clipping the corner.
3. Handling of the gel: At the end of electrophoresis, the gel needs to be carefully pried off the glass plate to avoid damaging the integrity of the gel. The pried-off gel needs to be equilibrated in the transfer buffer for a period of time to remove excess salts and other impurities.
4. The production of sandwich structure: transfer film, in accordance with the "filter paper - film - gel - filter paper" order to make the transfer film "sandwich" structure. In this step, it is necessary to pay attention to each layer should not have air bubbles, otherwise it will affect the effect of film transfer. You can use a glass rod to gently roll out the air bubbles.
4. Blocking
The transferred membranes were incubated on a shaker with 5% skimmed milk ( TBST preparation) at room temperature, and phosphorylated protein assay with 5% BSA ( TBST preparation) buffer, and closed at room temperature for 1-2 h. The main function of the sealant is to occupy non-target binding sites and reduce the background signal, thus increasing the specificity and sensitivity of the assay. Commonly used sealants: skim milk powder, bovine serum albumin (BSA).
1. Skimmed Milk Powder: Skimmed milk powder is a commonly used containment solution that is suitable for most experimental conditions and is relatively inexpensive. To ensure the best results, it is best to use it as it is prepared. It should be noted that skim milk powder contains a large amount of casein, which may produce a high background in the detection of phosphorylated proteins, when other containment solutions should be chosen. In addition, it cannot be used in biotin-labeled antibody systems due to its own biotin content.
2. Bovine Serum Albumin (BSA): BSA is another commonly used blocking solution and is particularly good at detecting phosphorylated proteins. It has a single component and is suitable for most cases, but it does not seal the Fc receptor.
When sealing non-phosphorylated proteins, it is more effective to use skim milk powder. Because BSA contains only one protein with a molecular weight of 66 KDa, while milk powder contains multiple proteins, it provides better containment.
Fig. 2. Effect of sealant on WB detection[3].
5. Antibody Incubation
(1) Incubation of primary antibody: dilute primary antibody (5% skimmed milk dissolved in TBST, phosphorylated proteins using 5% BSA dissolved in TBST) and incubate at 4°C overnight.
(2) Membrane washing: the membrane was washed according to 3 times TBST for 5 min each time.
(3) Incubate secondary antibodies: TBST-dissolved 5% skimmed milk, phosphorylated proteins using TBST-dissolved 5% BSA for 1-2 h at room temperature.
(4) Membrane washing: the membrane was washed according to 3 times TBST, 1 time TBS for 10 min each time.
How to select internal control?
Internal control antibodies play a crucial role in experiments, and to choose reliable internal antibodies, it is essential to adhere to the following three principles.
I. Sample sources and characteristics
1. Sample species and genus sources: GAPDH, β-Actin, etc. for mammalian samples; Plant Actin, Rubisco, etc. for plant samples. For less-studied sample types, relevant literature can be consulted for recommendations of suitable internal reference proteins. For example: Shiyang Li et al. found that the gatB gene is the most suitable endogenous gene for Mycoplasma pneumoniae in pigs[4].
2. Sample tissue characteristics: the structure and function of cells in different tissues vary, and proteins that are stably expressed in that tissue should be selected as internal references.
II. Localization of target proteins
According to the different localization of proteins, corresponding internal references have been sorted out, as summarized in the following table.
III. Molecular weight of target protein
To ensure that the target protein can be clearly distinguished from the internal reference protein, the molecular weight of the selected internal reference protein should differ from the target protein by at least 5 kDa. (To facilitate simultaneous incubation of the internal reference antibody and the antibody to the target protein on the same membrane to reflect the expression difference between samples more accurately). For example, when the molecular weight of the target protein is 45 kDa, preference may be given to either GAPDH or β-Tubulin. the molecular weight of GAPDH is approximately 36 kDa, which is sufficiently different from the 45 kDa target protein to be clearly distinguishable.
Tips
It is important to note that there is no one internal reference for all tissues and cells[5], here are some special cases are shown.
1. Tissue-specific internal reference selection
(1) Adipose tissue: the expression of β-Actin is very low and not suitable for use as an internal reference.
(2) Comparison of multi-tissue and multi-cell samples: as a metabolic protein, the expression of GAPDH is relatively constant in living tissues, so it is recommended to use GAPDH as an internal reference when comparing multi-tissue and multi-cell samples[6]. Structural proteins, such as β-Actin and β-Tubulin, will have expression differences in different tissues.
2. Selection of internal reference for the detection of modified proteins
Detection of modified proteins such as phosphorylation and acetylation: relatively stable expression of structural proteins such as β-actin and β-Tubulin.
3. Selection of internal references under disease or treatment conditions
(1) Hypoxia, diabetic models, tumor tissues: increased GAPDH expression, unsuitable for use as an internal reference[7].
(2) Anti-cancer and fungal drug treatment: Tubulin expression is affected and unsuitable as an internal reference.
4. Selection of internal references during changes in cell function or state
(1) Cell proliferation assays: c-Jun changes in its own expression and is not suitable as an internal reference[8].
(2) Apoptosis experiment: TBP and Lamin will change their expression or localization during apoptosis, and are not suitable as nuclear internal reference.
5. Selection of internal references for special organizational types
Skeletal, cardiac, and smooth muscle: Tubulin expression is altered and specialized, not suitable for use as an internal reference.
6. Internal reference selection for special sample types
Secreted samples such as plasma, breast milk, tissue fluids, etc.: Secreted proteins such as Transferrin can be selected as an internal reference due to the lack of intact cellular structure.
Tips of antibody incubation
1. In order to reduce non-specific binding, containment solution can be used as the antibody dilution solution for primary and secondary antibodies.
2. The primary antibody incubation conditions are recommended to be 4° overnight, and the secondary antibody incubation conditions are at room temperature for 1h.
3. TBST buffer is recommended for washing solution. Maintain a moderate shaking speed during the washing process to avoid excessive speed, which may result in the shedding of proteins or antibodies from the membrane, and to ensure that the washing solution can fully contact every corner of the membrane. During the washing process, replace the washing solution with a new one at the right time to ensure the washing effect.
4. It is necessary to keep the membrane moist during the washing process to avoid drying of the membrane leading to decreased antibody binding or protein denaturation.
6. ECL development
(1) Configure ECL Chemiluminescent Reagent Liquid A and Liquid B 1:1 and mix well in an environment protected from light.
(2) The PVDF membrane was placed on the carrier stage of the chemiluminescence imager.
(3) Pipette an appropriate amount of ECL mixture onto the PVDF membrane, making sure that the working solution evenly covers the entire blotting membrane.
(4) Push in the stage and set the exposure time for image acquisition (different exposure times can be set for image acquisition, from which the image with the best exposure effect is selected).
Although WB experiments seem simple, they contain a lot of learning. Only when we really master every detail and strictly follow the experimental steps can we obtain accurate and reliable results. We hope today’s sharing helps with your WB experiments. Finally, we wish scientists using MCE antibody products can publish high - scoring papers and secure loads of funding!
Actin is an important cytoskeletal protein found in essentially all eukaryotic cells. Its amino acid sequence is highly conserved and has changed little during evolution.
VDAC1 is an important transmembrane protein on the outer mitochondrial membrane that plays an important role in regulating cell survival and maintaining homeostasis of the intra- and extracellular environment.
LAMP2 is present in the lysosomal membrane and plays an important role in cellular autophagy, lysosomal function, and a variety of physiological and pathological processes.
