[Experimental technical notes] Western Blotting experimental operation points and data analysis

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1. Sample Preparation

• ① Lysis :
  ◆ Cells : Discard the culture supernatant , wash 1-2 times with sterile PBS (remove the residual medium on the surface of the cells and the culture dish, because the serum in the medium is rich in protein), add the cell lysate (80-100ul/ 60mm dish) and protease inhibitors , shake and lyse on a shaker , and observe at any time. It is best to lyse on ice (put an ice box on the shaker, and put the petri dish in the ice box).
  ◆ Tissue : Cut the tissue block into a suitable size , rinse with sterile PBS 1-2 times, add cell lysate and protease inhibitor, and homogenize with a high-speed homogenizer (low temperature homogenization: the outside of the 2ml flat-bottomed EP tube with the tissue block Set a small beaker filled with crushed ice) or high-throughput tissue crushing instrument for crushing (pre-cool the lysate in advance, divide the crushing process into several sections, shorten the crushing time of each small section, and put the EP tube containing the tissue in the broken gap Bury in crushed ice to cool for a few minutes before the next short crushing process). The best low temperature cracking .
• ② Remove debris (after fully lysing cells or tissues, there will be some cell debris that cannot be completely dissolved in the lysate and needs to be removed): centrifuge at 12000rpm for 10-20min at 4°C, and take the supernatant. (Cell debris sinks to the bottom of the tube after centrifugation, and the supernatant is the extracted protein)
• ③ Protein quantification : BCA method is used to determine the protein concentration and then aliquoted.
• ④ Protein denaturation : add loading buffer, boil in boiling water for 5 minutes. (Boiling water bath can easily flush the cap of EP tube. In order to prevent contamination, a metal bath or PCR machine can also be used to denature the protein.)
• ⑤ Storage : Aliquot and store denatured protein samples at -20°C, and long-term storage at -80°C. It can also be frozen at -80°C as soon as the lysis is completed, and the samples can be taken out after the samples are gathered together for subsequent steps such as de-fragmentation, quantification, and denaturation.
• Recommended reagents:
  △ 碧云天，Western 及 IP 细胞裂解液（P0013） Cleavage
  △ Pierce™ BCA Protein Assay Kit（23227） Protein concentration determination , recommended to use 96-well plate + microplate reader, save reagents (200ul BCA working solution per well), save trouble and time (scan the microplate reader, paste the data into excel, The concentration of each protein can be calculated, and the template can be saved for the first calculation, and the next time you can directly fill in the data in the template to get the concentration and the volume of each tube).
  BCA standard music production: (The operation of the official manual is more complicated, let’s simplify it) The concentration of the BSA standard that comes with the kit is 2ug/ul, and the gradient of the standard curve is set: prepare 7 wells, and add 20, 19, 18, 17, 16, 15 to each well in sequence , 14 ul deionized water, 0, 1, 2, 3, 4, 5, 6 ul BSA standard, so the obtained standard protein point is 0, 2, 4, 6, 8, 10, 12 ug (can cover common Lysate protein extraction range, super high concentration of protein is not applicable).
  
  △ Sigma，Sample Buffer, Laemmli 2× Concentrate（S3401） The protein is denatured , the protein sinks to the bottom well, it is not easy to float, and the sample is loaded smoothly. Disadvantages: 2×, so it is only suitable for samples with high protein concentration . Just ordinary protein extraction can also use the loading buffer of Biyuntian (domestic products).

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2. SDS-PAGE gel preparation and electrophoresis separation

2.1 Glue filling

• ① Washing glass plates : There are different specifications of glass plates, which can prepare PAGE gels of different thicknesses . Care should be taken to select suitable glass plates for glue making . Rinse with ionized water several times, and let the glass piece lean against the wire basket to dry naturally , taking care not to have water stains.
• ② Assembling the plastic frame : the glass plates should be paired (one is a thick plate with glass right angles , the other is a smooth thin plate ), align the paired glass plates, and keep the bottom edges of the two glass plates at the same level The glue-making bracket is fixed , and then installed on the fixing frame. Pay attention to the tightness (tightness) of the glue-making bracket, the elasticity (tightness) of the spring on the clip on the glue-making fixing frame, and whether the rubber strip on the fixing frame is clean and has good resilience . Always pay attention to whether there is leakage when pouring glue glue ,If there is a slight leak , you can clamp a 1ml pipette tip on the holder, as shown in the picture above, you can artificially tighten the clip on the holder. If the leak is severe , there is nothing to do, and the set can only be assembled again . soIt is best to match several pieces at the same time , there is always one that can be used。
• ③ Glue preparation : The PAGE gel used in western is discontinuous and divided into two layers , the upper layer is stacking gel , and the lower layer is separating gel . When mixing, mix the lower layer of separating gel first , so thatPress the glue with 500ul absolute ethanol (better than water pressure), After the separation gel is solidified, pour off the absolute ethanol, and after the ethanol volatilizes, pour in ionized water to clean the residual ethanol, and then match the upper layer of laminating gel. The stacking gel needs to have a certain height. Generally, the distance between the bottom of the sample well and the separation gel should be 1-2cm . If the distance is too short, the pressure will not be good. If the distance is too long, the separation gel will be short, which will affect protein separation.
• ④ Comb insertion : Combs have different specifications , which are suitable for different thicknesses of glue and sample volumes, so attention should be paid. Insert the comb immediately after pouring the layered glue , and the action should be fast, otherwise the sample hole will be easily deformed. When inserting the comb, it is best to use a piece of thick straw paper to block it, because unsolidified acrylamide is poisonous , and it is easy to spray the face when inserting the comb, so pay attention to protection . Polyacrylamide that is completely polymerized into a gel is much safer. So when discarding the remaining glue, you can wait for it to completely solidify before discarding it . Sometimes when the comb is unplugged and the space between the sample holes is unsteady, it must be that the action of inserting the comb is slow.
• ⑤ Pull out the comb : Don’t pull out the comb in a hurry after the laminating gel is solidified , but remove the set of glass plate-glue-comb as a whole , put it into the electrophoresis tank, pour the electrophoretic solution, and then pull it out . The electrophoretic solution contains SDS, it will be smoother when pulling out the comb.
• SDS-PAGE glue formula:
  △ The last in the table X%is a general calculation formula , adjust the amount of ddH ₂ O with the amount of 30% acrylamide .
  △ 10% AP and TEMED are catalysts that catalyze the polymerization of acrylamide . TEMED can be stored at 4°C for a long time ; Ineffective, so after buying it back, prepare it directly, then pack it into a 500ul tube, freeze it at -20°C, take out a tube to dissolve when you use it, and use it up within a week.)
  

2.2 Electrophoretic separation

• Principle : The sample will be compressed into a thin line in 5% stacking gel , which is the effect of glycinate ions Gly ^- and Cl ^- in the buffer . In the stacking gel , Cl ^- runs at the front, the protein sample in the middle, and the Gly ^- salt ion at the end. A voltage gradient is formed between Gly ^- and Cl ^{- to compress the protein sample; when it reaches} the separating gel , Gly ^- , Cl ^-Running in front of the protein, the protein sample is out of the limit of the voltage gradient and begins to swim at different speeds according to the size of the protein, thereby separating it according to the size of the protein.
  
• Laminated gel concentration : about 5%, pH 6.8.
• Separating gel concentration : 7.5-15%, pH 8.8. Different concentrations of separating gels have different separation abilities for protein samples. The lower the concentration , the better the separation, but small proteins are easy to escape; 10-12% is commonly used.
• Precautions for electrophoresis:
  ① SDS-PAGE is sensitive to ions. Use deionized water to prepare the electrophoresis solution . The electrophoresis tank and the container with the electrophoresis solution also need to be rinsed with deionized water .
  ② Before loading the sample, flush the sample hole with an insulin needle . ③ Prepare the electrophoresis solution one day in advance, mix well and put it at 4°C . When it is time to use, the electrophoresis tank is slightly tilted, and the electrophoresis solution is poured slowly along the inner wall of the tank. It can ensure that there are no air bubbles at the bottom edge of the PAGE gel . (SDS is contained in the electrophoresis solution. If it is prepared and used immediately, the foam produced by SDS will affect the electrophoresis, such as accumulating on the bottom of the PAGE gel, resulting in differences in each lane.) ④ Slowly add protein samples to the wells , and the top of each well It is best to keep the sample volume consistent. The well without protein is also added to the same volume with loading buffer, so that the protein bands that come out can be more uniform, otherwise the protein bands will float to the small or empty well. . Don't forget to put a pre-stained protein marker on the well .
  
  

• Recommended reagents :
  △ 天能(domestic high-quality products), SDS-PAGE gel-making bracket, gel-making fixed frame, comb, glass plate
  △ If you don’t want to prepare the mother solution and electrophoresis solution for the gel, it is recommended 迪生(the product quality is stable) SDS-PAGE stocking bufferand TGS buffer.
  △ Pre-stained protein markers can be Thermo Scientificused PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa（26619）.

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3. Transfer film

• There are two kinds of wet transfer and semi-dry transfer .
  Wet transfer : soak all in a large amount of transfer solution. The sandwich is vertically inserted into the transfer film tank, from the negative electrode to the positive electrode: sponge pad, filter paper, glue, PVDF membrane , filter paper, sponge pad.
  Semi-dry transfer : A small amount of transfer solution keeps the sandwich moist. The semi-dry transfer instrument is horizontal, with the negative pole on the top and the positive pole on the bottom. There is no sponge pad, and the filter paper is directly attached to the electrode plate. From the positive pole to the negative pole, the order is: positive metal plate, filter paper, PVDF membrane, glue, filter paper, negative metal plate, plastic cap.

• Sandwich making and transfer solution formula for wet transfer and semi-dry transfer :
  

• recommend:GE，PVDF 膜（A10074129）

• Notes for membrane transfer:
  ① It is recommended to use PVDF membrane (high protein-binding efficiency), because the efficiency of NC membrane-binding protein is about 80-100ug/cm ² , while PVDF membrane can reach 155-300 ug/cm ² .
  ② When transferring the membrane, prepare 2 plates , the large plate is poured with the transfer solution , and the small plate is poured with an appropriate amount of methanol . Peeloff the glue from the glass plate, cut off the part of the laminated glue , and then soak it in the transfer solution. The filter paper of the same size as the glue is also soaked in the transfer solution. Don’t rush to soak the cut PVDF membrane in methanol, but let it float gently on the surface of the methanol liquid for 10s . During these 10s, methanol is enough to activate the PVDF membrane , and attention should be paid to the activation of the PVDF membrane. We can't kill it at the end, so we can start making sandwiches first,When the PVDF membrane is to be added, throw the membrane into methanol to activate, immediately loaded into sandwiches as soon as activated.
  ③ When making sandwiches, you need to use a glass rolling rod, or take a clean and smooth glass test tube to grind away the air bubbles between the membrane and the glue .
  ④ Wet transfer conditions are generally set at 10:00 pm 200mA 恒流，2.5-3h, the time is relatively long, and heat generation needs to be controlled , because heat generation will affect the film transfer effect. It is best to prepare a large ice box filled with crushed ice, add some water, and bury the transfer film tank in the ice-water mixture to cool down ; you can also add a magnetic rotor to the transfer film tank, and put the ice box and the transfer film tank in a magnetic On the stirrer , stir again and turn; or directly put the transfer tank into a 4°C freezer to cool down .
  Bole ’s transfer film tank is very hot and needs to be controlled; Pharmacia ’s transfer film tank has better heat control and can achieve no heat at all.
  ⑤ The semi-dry sandwich should be kept moist, but the metal plate around the sandwich should be kept dry .
  ⑥ The condition for semi-dry transfer is to use 恒压 20V，20min-1h. If the protein is large, the transfer time can be extended.
  ⑦ Wet transfer can transfer a wide range of protein sizes . Ordinary semi-dry transfer is suitable for transferring proteins of 30-120KD size. Some specially designed semi-dry transfers can transfer proteins of any size just like wet transfer. This depends on the specific machine.
  ⑧ Deionized water is also required for transfer solutionFor preparation, the transfer tank and container need to be rinsed with deionized water,The transfer solution formula needs to be adjusted according to the protein size, large protein ( >100 kD): SDS 1g, methanol 0 ml; small protein ( <100kD): SDS 0g, methanol 200ml.
  ⑨ In the process of membrane transfer, the effects of SDS and methanol are just opposite.
  · After SDS is combined with protein, it can make the protein transfer out from the gel more easily , but it can also prevent the protein from binding to the membrane , especially the binding of the protein to the NC membrane. Therefore, large proteins are not easy to transfer out, and additional SDS should be added; small proteins can be transferred out by SDS in PAGE gel. Excessive SDS will also affect the current and increase heat, thus affecting the antigenicity of some proteins. Therefore, it is necessary to control the content of SDS according to the situation.
  · Methanol can destroy the combination of SDS and protein and promote the combination of protein and membrane , but it will reduce the pore size of PAGE gel and cause some proteins to precipitate, thus affecting the experimental results, so the amount must be strictly controlled .

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4. Antibody hybridization

• 1> Blocking : use 5%脱脂牛奶or 5% BSA( TBSTdissolved with) as blocking solution , blocking conditions : 室温 1h，或 4℃过夜. (After the target protein is transferred to the membrane, but there are many other proteins on the membrane, because the total protein is used for running the gel, other non-target proteins will also be transferred to the membrane, which will expose many antibody binding sites, so The membrane needs to be blocked before antibody hybridization.)
  ◆ BSA : It is generally used for the detection of protein phosphorylation modification , because milk is rich in phosphatase, which will change the state of protein phosphorylation modification.
  ◆ Sometimes, the blocking solution formula and blocking conditions also need to be changed, especially for large proteins, 5% skimmed milk is not enough to block for one hour; some proteins may not be used with milk and BSA, you can use gelatin to block or directly apply antibodies without blocking.

• 2> Primary antibody incubation : dilute the primary antibody with blocking solution , overnight at 4°C, or at room temperature or 37°C for 1-X h.

• 3> Wash the membrane with TBST , room temperature, 5min x 4 times.

• 4> Secondary antibody incubation : 1h at room temperature.

• 5> Wash the membrane with TBST , room temperature, 5min × 8-12 times.

• 6> ECL color development , X-ray film pressing or film scanning.

• TBS formula : just
  1L TBSadd in . : A non-ionic detergent, which has the function of refolding antigen, which can improve the specific recognition ability of antibodies; improve the blocking effect, and block the unbound sites on the membrane with inert protein or non-ionic detergent, which can reduce the antibody Non-specific binding; does not destroy protein structure when emulsifying protein.1ml Tween 20TBST
Tween 20
  

• Recommended reagents :
  Dickson , TBS. (One packet of powder contains 500mL)
  GE , Amersham ECL Prime Western Blotting Detection Regent (RPN2232) are effective, but expensive, and Biyuntian has a higher cost performance.
  Thermo, SuperSignal West Pico Chemiluminescent Substrate (34080).

5. Image Analysis

• Use Gel-Pro Analyzer or ImageJ (recommended)
• Reference:
  Basic scientific research for clinicians - 13. WB result processing (recommended to read this)
  [Experimental technology study notes] Western Blot band analysis｜Image J and Graphpad band grayscale analysis and processing process
  Western blot experience inventory, here is One article is enough
  to measure electrophoretic pictures with professional-grade Gel-Pro

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6. Common problem analysis

1) Non-specific bands:

• Antibody concentration is too high: ◆ Decrease antibody concentration
• Insufficient blocking:
  ◆ Increase blocking solution concentration, e.g. from 5% to 7%
  ◆ Increase blocking time and/or temperature
  ◆ Add 0.05% Tween 20 to blocking buffer solution
  ◆ Use same blocking solution for antibody dilution
• SDS causes non-specific binding of antibodies and protein bands:
  ◆ Wash the blot after transfer.
  ◆ Do not use SDS during immunoassay.
• Antibody Quality: Try Different Antibodies

2) Weak or no banding

• Antibody problems, low activity or low concentration, low affinity primary antibody, mismatched primary and secondary antibodies:
  ◆ Increase antibody concentration
  ◆ Antibody may have lost activity, perform dot blot to determine its activity and optimal concentration
  ◆ Tests are different Antibody and/or primary-secondary combination
• Insufficient antigen-antibody reactivity:
  ◆ Increase total protein loading per sample in the gel
  ◆ Check sample integrity to ensure protein is not degraded
• Antigen blocked by blocking solution
  ◆ Try a different blocking solution (e.g., avoid milk when detecting phosphorylated proteins)
  ◆ Optimize blocking solution concentration, 3–5% BSA or nonfat dry milk is recommended
• Poor performance of chemiluminescent substrate
  ◆ Increase substrate incubation time
  ◆ Prepare a small amount of working solution to determine whether the substrate has lost activity. In a dark room, add a small amount of HPR conjugate to the substrate working solution and blue light should appear. If not, the substrate or HRP conjugate must be inactive.
  ◆ Make sure there is no cross-contamination between the two bottles in the substrate kit. If cross-contamination occurs between the two reagents, the activity of the reagents will decrease.
  ◆ Azide is an inhibitor of HRP, do not use azide in the secondary antibody buffer
• Antibody removal and rehybridization on blot
  Optimize antibody removal conditions
  Perform rehybridization detection only when necessary
  Avoid repeated rehybridization of the same blot
• Insufficient transfer
  ◆ The transfer time is too short. If wet transfer is used, it is necessary to increase the transfer time.
  ◆ The transfer electric field is too weak. Increase the transfer voltage or current.
  ◆ The transfer solution is not optimized. Add 0.01- 0.05% SDS
  ◆ Reduce the concentration of methanol in the transfer buffer to 5% or less.
  ◆ The gel selection is not appropriate, use a lower percentage of polyacrylamide gel
  ◆ Use ponceau red staining solution to stain the membrane and Coomassie brilliant blue staining solution to stain the gel after transfer to confirm the transfer efficiency, and optimize the transfer on this basis Membrane condition.
• Transferred
  ◆ Transfer time is too long, reduce time
  ◆ Transfer electric field is too strong, reduce transfer voltage or current
  ◆ Transfer solution is not optimized, equilibrate the gel in the transfer solution for 20 minutes before assembling the “sandwich” to reduce Increase the concentration of SDS in the gel to avoid excessive transfer of small molecular weight proteins.
  ◆ Increase methanol concentration in transfer buffer to 40%
  ◆ Inappropriate gel selection, use higher percentage acrylamide gels
  ◆ Use Tris/tricine gels when target protein molecular weight is less than 10 KD. Use a 0.2 µm membrane to resolve overtransfer of small proteins.
  ◆ Small proteins will pass through the large-pore blotting membrane, and a 0.2 μm membrane is superimposed on the 0.45 μm membrane to detect the excessive transfer of small proteins

3) The signal is oversaturated

• Hollow bands:
  ◆ Loading volume is too high, reduce total protein loading per sample
  ◆ Too many antibodies, reduce primary antibody and/or secondary antibody concentration
  ◆ Chemiluminescent substrate is too sensitive, replace with less sensitive chemistries Luminescent substrate
• Bands are so dense that bands stick together:
  ◆ Load too much sample, reduce total protein load per sample
  ◆ Too much antibody, reduce primary and/or secondary antibody concentration

4) Bubbles on strip:

• Improper sandwich assembly
  ◆ Use more transfer buffer when assembling the “sandwich”
  ◆ Roll out all air bubbles between the gel and the membrane when assembling the “sandwich”
  ◆ Use Ponceau before antibody incubation Stain the membrane with dye solution to check the quality of the transfer membrane.

5) Uneven film transfer:

• Partial drying or uneven hydration of the blot
  Make sure the entire blot is evenly submerged and equilibrated in transfer buffer
  PVDF membranes need to be pre-wet equilibrated with 100% methanol before placing in the buffer
• Hardware problems
  ◆ Check the wiring connections of the wet transfer unit
  ◆ Make sure the metal plate is clean and free of physical damage, even slight bending of the metal plate can drastically change the electric field strength of a semi-dry transfer
  system The uniform electric field of the film instrument has technical content, so the money for buying a film transfer instrument cannot be saved)

6) The background is too strong:

• Primary and/or secondary antibody concentration too high: dilute primary and/or secondary antibody concentration
• Insufficient blocking
  ◆ Increase blocking buffer concentration, e.g. 3–5% bovine serum albumin, casein, skim milk
  ◆ Decrease blocking time and/or temperature
  ◆ Add 0.05% Tween-20 to blocking buffer
  ◆ Use same antibody diluent Blocking solution (add 0.05% Tween-20)
• Wrong blocking solution used, primary and/or secondary antibody bound to protein in blocking buffer: try another blocking solution (albumin, casein, skim milk, etc.). Do not use milk to block blots when using the avidin-biotin system—milk contains biotin
• Insufficient washes
  ◆ Increase the number of washes and buffer volume (minimum 5 × 5 minutes)
  ◆ If the wash buffer does not contain
• Exposure time is too long: reduce imaging exposure time
• The blot dries out during blotting
  Make sure the blot stays wet
  Make sure the blot is kept covered with enough liquid to prevent it from drying out
• Buffer reuse leads to contamination: use fresh buffer

7) The background is smudged

• Part of the blot dries out
  Make sure the blot stays wet
  Make sure the blot is kept covered with enough liquid to keep it from drying out
• Insufficient wash buffer contact with some blot areas
  Increase wash buffer volume
  Avoid stacking too many blots in one incubation vessel
• Insufficient contact of wash buffer with some blot areas: use a clean or new transfer "sandwich"
• Contamination of the blot
  ◆ Do not touch the blot directly with your hands. Always wear cleaning gloves or use medical tweezers
  Ensure electrophoresis equipment, transfer equipment, and incubation plates are clean and away from external sources of contamination

8) The background is speckled

• Gel fragments adhere to the blotting membrane: Remove the acrylamide gel fragments remaining on the surface of the blotting membrane
• Clumped blocking agent adheres to the blot
  ◆ Make sure the blocking agent is completely dissolved before use
  ◆ Add 0.1% Tween-20 to the blocking buffer solution
• Antibody aggregation
  ◆ Use 0.2 μm filter to filter the antibody buffer solution
  ◆ Use fresh antibody
  ◆ Add 0.1% Tween-20 to the antibody buffer solution
• Buffer contamination
  ◆ Use new buffer
  ◆ Filter buffer with 0.2 μm filter

9) The content of internal reference protein is inconsistent among different samples

• During the experiment, the expression level of the housekeeping protein selected as the internal reference changes
  . Identify and verify the consistency of the selected internal reference protein under the experimental conditions, which means that the current experimental conditions will not change the expression level of the internal reference protein. Use a dye to
  give The total protein on the membrane was stained, and the total protein detected on the membrane was used as an internal reference

10) Internal reference protein detection signal saturation

• During the experiment, the expression level of the housekeeping protein selected as the internal control changes
  . Reduce the sample volume to eliminate signal saturation
  . Dilute the antibody concentration or reduce the incubation time to avoid detection signal saturation
  . Select another low-abundance protein with consistent expression as Internal reference
  Stain the total protein on the membrane with a dye, and use the total protein detected on the membrane as the internal reference