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Western Blot Tips and Tricks!
January 21, 2025
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Overview
Western blotting (WB) is a method that transfers proteins onto a membrane and detects them using antibodies. For known expressed proteins, specific antibodies can be used as primary antibodies for detection. For the products of novel gene expression, detection can be achieved through antibodies against fusion tags. Similar to Southern or Northern blotting, Western blotting employs polyacrylamide gel electrophoresis, with proteins as the target molecules, antibodies as probes, and labeled secondary antibodies for visualization.
Protein samples separated by SDS-PAGE are transferred onto a solid-phase carrier (e.g., nitrocellulose membrane), which adsorbs proteins non-covalently and maintains the peptide types and biological activities as separated by electrophoresis. The proteins or peptides on the solid-phase carrier act as antigens, triggering an immune reaction with corresponding antibodies, followed by a reaction with enzyme- or isotope-labeled secondary antibodies. Detection is achieved through substrate color development or autoradiography to identify the specific protein components of gene expression. This technique is also widely used for detecting protein expression levels.
Western blotting is a complex and multi-step procedure, increasing the likelihood of unintentional errors by researchers, which significantly affects its accuracy and reproducibility. In terms of operational difficulty, WB ranks among the most challenging laboratory techniques. Below are the steps for WB and recommended products.
I. Reagent Preparation
1) 10x Electrophoresis Buffer: 30.3 g Tris Base, 144 g Glycine, and 10 g SDS. Measure 800 mL ddH2O, dissolve thoroughly, and adjust the volume to 1 L;
2) 10x Transfer Buffer: 30.3 g Tris Base, 144 g Glycine. Measure 800 mL ddH2O, dissolve thoroughly, and adjust the volume to 1 L;
3) 1x Electrophoresis Buffer: 100 mL of 10x electrophoresis buffer, adjust the volume to 1 L with water;
4) 1x Transfer Buffer: 100 mL of 10x transfer buffer, 200 mL methanol, adjust the volume to 1 L with water;
5) 10x TBS: 60.6 g Tris-base, 87.66 g NaCl. Measure 800 mL ddH2O, dissolve thoroughly, and adjust the volume to 1 L;
6) 1x TBST: 100 mL of 10x TBS, adjust the volume to 1 L with water, add 1 mL Tween-20;
7) 10% SDS Solution: Dissolve 10 g SDS in 100 mL ddH2O and store at room temperature;
8) Blocking Buffer: Dissolve 5 g skimmed milk powder in 100 mL 1x TBST to prepare a 5% blocking milk solution.
Researchers can directly order products from Absin to save time for scientific research!
II. Protein Sample Preparation
Extraction of Total Cellular Protein
1) Discard the culture medium and aspirate any remaining liquid with a pipette;
2) Wash the cells with 3 mL of pre-chilled PBS (4℃) per flask. Gently shake and discard the PBS. Repeat the washing process three times;
3) Prepare RIPA lysis buffer by adding 10 µL of PMSF (100 mM) to 1 mL of RIPA buffer, mix well and place on ice;
4) Add 400 µL of the lysis buffer containing PMSF to each flask and lyse the cells on ice for 30 minutes. Frequently agitate the flask to ensure complete lysis;
5) After lysis, scrape the cells to one side of the flask using a cell scraper, then transfer the cell debris and lysate to a 1.5 mL centrifuge tube;
6) Centrifuge at 12,000 rpm for 5 minutes at 4℃ (pre-cool the centrifuge before use);
7) Aliquot the supernatant into centrifuge tubes and store at -20℃.
III. Protein Quantification
Preparation of Standard Curve and Detection of Sample Protein Concentration
We recommend the Absin BCA Protein Assay Kit (abs9232). Follow the instructions provided in the manual strictly.
IV. SDS-PAGE Electrophoresis
Gel Preparation
Prepare the resolving gel by adding solutions in sequence, mixing thoroughly after each addition. Seal the resolving gel with water after preparation, and allow it to polymerize for at least 40 minutes. Then add the stacking gel, insert the comb without generating bubbles, and allow it to polymerize for 30 minutes. Avoid dislodging the comb. This is the traditional method of gel preparation, which is both time-consuming and labor-intensive. We recommend Absin's precast gel products to save time.
Product Features:
1) Utilizes automated gel casting technology to ensure high stability and reproducibility of product quality;
2) Uses glass plates to effectively reduce non-specific protein adsorption, resulting in sharper and clearer protein bands;
3) The gel holder can be easily opened by simply scratching one side with a blade;
4) The gel does not contain SDS and can be used for both denaturing and non-denaturing electrophoresis;
5) Compatible with mainstream mini-gel tanks on the market, such as Bio-Rad, Invitrogen, Thermo Fisher, and Junyi Oriental;
6) Short electrophoresis time. The recommended electrophoresis voltage and time for this precast gel are 150 V for 40-50 minutes, which can complete the electrophoresis and produce very flat and sharp bands.
V. Loading and Electrophoresis
1) Sample preparation: Heat the protein samples at 95℃ for 10 minutes, vortex briefly, and centrifuge. Allow to cool to room temperature;
2) Assemble the gel cassette in the electrophoresis apparatus, ensuring that the shorter glass plate faces inward and is tightly clamped without leakage;
3) Fill the electrophoresis tank with electrophoresis buffer until the entire apparatus is filled;
4) Load the protein samples and marker into the wells according to the protocol. Use 3 µL of marker;
5) Close the lid, ensuring correct orientation, and connect to the power supply. Electrophoresis is completed at 150 V for 40-50 minutes.
VI. Electrotransfer
1) PVDF membranes need to be activated in methanol;
2) After electrophoresis, remove the gel cassette with the shorter glass plate facing up. Remove excess gel and carefully lift the gel off the longer glass plate in water;
3) In the transfer buffer, place the transfer sandwich in the following order: sponge mesh - filter paper - gel - PVDF membrane - filter paper - sponge mesh. Ensure the PVDF membrane is in close contact with the gel and remove any bubbles. Adjust the transfer cassette with the black side facing the negative electrode and the white side facing the positive electrode. Fill the transfer tank with transfer buffer and transfer the proteins at a constant current of 200 mA for 120 minutes (conditions may need to be optimized). Surround the transfer tank with ice to maintain low temperature.
VII. Blocking
1) After transfer, the marker should be completely transferred from the gel to the membrane;
2) Quickly rinse the membrane with TBST to remove residual transfer buffer;
3) Immerse the PVDF membrane in 5% blocking milk and incubate with gentle shaking at room temperature for 1 hour.
VIII. Antibody Incubation and Chemiluminescent Imaging
1) After blocking, wash the membrane with 1x TBST buffer to remove excess milk. Wash the membrane for 5 minutes each time, for a total of 3 times. Dilute the primary antibody in primary antibody dilution buffer or TBST at a ratio of 1:5000 (conditions may need to be optimized). Incubate the membrane in the primary antibody solution overnight at 4℃ with shaking;
2) After primary antibody incubation and recovery, wash the membrane with 1x TBST buffer on a shaker for 5 minutes, for a total of 3 times. Dilute the secondary antibody in 5% skimmed milk at a ratio of 1:1000 and incubate at room temperature with gentle shaking for 1 hour;
3) After secondary antibody incubation, recover the secondary antibody and wash the membrane with 1x TBST for 5 minutes, for a total of 3 times;
4) After washing, apply the chemiluminescent substrate to the membrane and expose it using a chemiluminescent imager. Analyze the grayscale using ImageJ software.
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Catalog No. |
Product Name |
Specification |
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Rabbit anti-Claudin 4(pY208) Polyclonal Antibody |
50uL |
|
|
Rabbit anti-DDOST Polyclonal Antibody |
50uL |
|
|
Rabbit anti-MLXIPL Polyclonal Antibody |
50uL |
|
|
Rabbit anti-MLXIPL Polyclonal Antibody(C-term) |
50uL |
|
|
Rabbit anti-Elk1 Polyclonal Antibody |
50ug |
|
|
Rabbit anti-GCLC Polyclonal Antibody |
100uL |
|
|
Rabbit anti-Heparanase Polyclonal Antibody |
50uL |
Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us
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Absin Bioscience Inc. |
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