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This is an easy tutorial about western blot from Elabscience, this video provides tips to help you learn how to prepare samples, electrophoresis, transfer your proteins from the SDS-PAGE gel onto a PVDF, block the membrane,add the primary and secondary antibodies etc.
Procedure:
1、Samples preparation.
RIPA preparation: Prepare RIPA or NP40 buffer supplemented with fresh protease and phosphatase inhibitors. Prepare when needed. Calculate the total volume of RIPA buffer before processing the samples.
Tissue Processing: Cut the tissues into pieces on ice as quickly as possible to prevent degradation by proteases. Add 200-300ul of ice-cold RIPA lysis buffer for about 5mg of tissue samples, and homogenize on ice. Centrifuge at 12000rpm for 5 minutes at 4°C, then take the supernatant.
Adherent cell processing: Take out the culture medium and wash it with ice-cold PBS. Add 150uL of prepared lysis buffer for per 5×10^6 cells, then crack the mixture on ice for 5-10minutes and mix it with scraper. Take the disrupted sample into an EP tube. Centrifuge at 12000rpm for 5 minutes at 4°C, then take the supernatant.
Suspension cell processing: Transfer the cells into a pre-cooled centrifuge tube. Centrifuge at 2000-3000rpm for 5 minutes at 4°C, then discard the supernatant. Add ice-cold PBS to wash the cells. Centrifuge at 2000-3000rpm for 5 minutes at 4°C, discard the supernatant. Add 100-150ul of ice-cold RIPA lysis buffer for each 5×10^6 cells, then put the mixture on ice for 5-10minutes and mix it with pipette. Centrifuge at 12000rpm for 5 minutes at 4°C, then take the supernatant.
2、Protein concentration Measurement
Standard curve : Dilute the BSA with PBS. The recommended concentrations of BSA are 0, 0.2, 0.4, 0.6, 0.8, 1mg/ml. A duplicate is recommended for each concentration.
Dilute the tested samples with PBS to detect concentration.
Add 200uL of BCA reagents to the standard and sample wells, store out of light for 20-30 minutes at 37℃.
Determine the OD value at 568nm with microplate reader.
Calculate the protein concentration according to the average OD value of samples with the equation. Then calculate the loading volume of samples according to the protein concentration. The recommended total protein amount of each sample is 50ug.
Once you have determined the concentration of each sample, you can freeze them at -20°C or -70°C for later use or preparing for immune precipitation or loading into a gel.
3、Sample denaturation
Use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS) to denature the sample, then heat the mixture at 95–100°C for 10 minutes.
Dilute the sample supernatant with 5×SDS buffer at the ratio of 4:1. The best loading sample volume is 10ul and you must ensure that the loading volume is similar among the samples, so the sample should be diluted with PBS if the concentration is too high.
Heating in boiled water for 10 minutes.
Store the processed samples at -20℃(validity for 6 months), or at -70℃ for long-term storage.
4、SDS-PAG Gel preparation
Pour the mixed separating gel into the gel tank between two glass panes to about 50px below the top of the plastic holder.
Inject anhydrous ethanol or ddH2O above it to avoid affecting polymerization.
Incubate at 37℃ for 1 hour till it polymerizes completely, then pour out the ethyl alcohol.
Wash with ddH2O.
Absorb with filter paper, avoid touching the gel.
Add the stacking gel
Insert the comb carefully and avoid of forming bubbles.
Incubate at 37℃ for 1 hour till it polymerizes completely, then take out the comb.
The gel can be stored at room temperature for about 1 week in a sealed container with soaked towels to avoid of desiccation.
5、Electrophoresis
Add some electrophoretic buffer to the electrophoresis chamber, then fix the gel into the electrophoresis chamber. Make sure that there is no bubble on the platinum wire at the bottom of electrophoresis chamber.
Add electrophoretic buffer to the electrophoresis chamber. The buffer must be above the sample wells and the bottom of the gel must be immersed by buffer. Rinse all wells with electrophoretic buffer.
Add samples according to the calculated loading volume and add the protein marker.
Electrophoresis--The electrophoretic voltage of stacking gel is suggested to be 80V, lower than the electrophoretic voltage of separating gel which is suggested to be 110-150V generally in inconsecutive system/1. Samples should be at the same level when it reaches the separating gel. Electrophoresis time is about 2-3 hours till the bromophenol blue reaches the bottom of gel.
6、Electrotransfer (Wet transfer)
Take out the gel after the electrophoresis finished.
Cut the gel and wash it with ddH2O to remove the electrophoresis salts and detergents.
Prepare filter papers.
Soak the PVDF membrane in methanol for 5 seconds to re-activate it.
Use tweezers instead of fingers to avoid of touching the membrane.
Soak the PVDF membrane and filter paper and fiber mats into the electro transfer buffer.
Make sure that the PVDF membrane was cut into the same size as the gel. Large overhangs may prevent a current from passing through the membrane.
Put the related appliances in the following order, black plate - fiber mats - filter paper - gel -PVDF membrane - filter paper - fiber mat - white plate. After sandwiching the gel and membrane between filter papers, clean out the air bubbles between the gel and membrane with a roller, pipette or 15mL EP tube. Or you can assemble the sandwich device in transfer buffer to avoid of forming bubbles.
In a wet transfer device, the gel and membrane are sandwiched between sponge and filter paper and all these are tightly clamped together to ensure that no air bubbles will form between the gel and membrane. The sandwich device is submerged in transfer buffer in an electrical chamber. The negative-charged proteins will move towards the positively-charged electrode, so as to bind with the membrane.
Put the electrical chamber in an ice bath. Make sure that the PVDF membrane is near the positive pole.
Set a constant current. The electric current of each chamber is suggested to be 150-300mA. Adjust the transferring time according to molecular weight of protein. The ratio of SDS, methanol in the transfer buffer, protein size, and gel percentage may affect the transfer efficiency.
7、Blocking
Block the membrane to prevent the non-specific binding of the primary and secondary antibodies with the membrane.
Take out the PVDF membrane after transferring finished. Blot the transfer buffer up with filter paper.
Put the membrane into a blocking implement. Add either 5% skimmed milk -TBST solution or 5% BSA- TBST solution to block the membrane.
Milk is cheaper but is not recommended for studies of phospho-proteins. Milk contains casein, which is a kind of phospho-protein, which will cause high background because the phospho-specific antibody can detect the presence of casein in the milk.
Some antibodies produce a stronger signal on me membranes blocked with BSA opposed to milk for unknown reasons. Check the application notes on the datasheet in case there are specific instructions on how to block the membrane.
Incubate on the shaker for 2 hours at room temperature.
Membranes can be stored in TBST at 4℃ for up to 1 week.
8、Primary antibody Incubation
Take out the PVDF membrane after blocking. Blot the transfer buffer up with filter paper.
Add diluted primary antibody at the suggested dilution to the blocked blotting membrane. Too much antibody will result in non-specific bands. The primary antibody can be diluted with 5% milk-TBST.
The incubation time can vary from a few hours to overnight (rarely more than 18 h), which depends on the binding affinity of the antibody with the protein and the abundance of protein. We recommend a higher dilution ratio of antibody and prolong the incubation time to ensure the specific binding. If incubating overnight in blocking buffer, it is imperative to incubate at 4°C, otherwise contamination will occur and thus destruction of the protein will happen because of bacterial infection especially for phospho groups.
9、Membrane Washing
Remove the blocking buffer. Wash with TBST for 2 minutes to remove the residual primary antibody.
Wash the membrane with washing buffer on shaker for 5 minutes. Repeat the washing 3 times.
10、Secondary antibody Incubation
Take out the PVDF membrane after washing, blot the washing buffer up with filter paper.
Add the diluted peroxidase conjugated secondary antibody at the suggested dilution, and optimize the dilution according to the results. Too much antibody will result in non-specific bands. You may incubate the secondary antibody in blocking buffer, but a reduction in background may occur at the cost of a weaker specific signal, which presumably because the blocking protein hinders the binding of the antibody with the target protein.
Incubate the secondary antibody on shaker for 2 hours at room temperature.
We recommend the horseradish peroxidase (HRP)-conjugated secondary antibodies because Alkaline phosphatase (ALP)-conjugated secondary antibodies are less sensitive.
11、Membrane Washing
Remove the blocking buffer. Wash with TBST for 2 minutes to remove the residual secondary antibody.
Wash the membrane with washing buffer for on shaker for 5 minutes. Repeat the washing for 3 times.
12、Detection
The detection method depends on the conjugation of secondary antibody. ECL and DAB are usually used.
For HRP-conjugated antibodies, enhanced chemiluminiscence (ECL) kit is generally used as substrate
ECL preparation: Mix the substrate A and B at the ratio of 1:1.Prepare when needed.
Take out the PVDF membrane, and blot the washing buffer up with filter paper.
Cover the blotting membrane with mixed substrate for 1-5 minutes, and then observe the fluorescence in darkness with detection machine.
The camera detects the chemiluminescence emanating from the membrane, then transfer the signal into a digital image for rapid analysis with software provided with the detection machine.
Also we can use X-ray film to fixed the images or use DAB as chromogenic substrate when we use Alkaline phosphatase (ALP)-conjugated secondary antibodies.
13、Data analysis
Analyze the bands with Bandscan software.
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