1. Use A Lysis Buffer Containing 1% SDS
Use a lysis buffer containing 1% SDS when preparing whole cell lysates to ensure membrane and other hard to solubilize proteins, even synaptic junction proteins, are completely solubilized.
1% SDS lyses everything, preserving proteins from protease and phosphatase enzymes. Additional cocktail inhibitors are not necessary when 1% SDS is used.
2. Add Fresh BME To Your Sample Buffer
When preparing lysate samples use fresh BME in 4X sample buffer to ensure samples are properly reduced and denatured.
BME reduces the sulfhydryl groups in proteins and is highly volatile. We recommend adding fresh BME to 4X sample buffer just prior to adding to your samples.
3. Visualize The Proteins On Your Blots
Stain your membrane with Ponceau S after transfer to confirm successful protein transfer and to determine the exact alignment of lanes and placement of the proteins on the gel.
Ponceau S will show you where protein has / has not transferred onto the membrane and allow you to visualize any areas where bubbles or other factors may have affected the transfer of protein from the gel to the membrane.
Make sure to collect excess Ponceau S for reuse and to thoroughly rinse membranes in dH2O after staining to remove any remaining excess Ponceau S. Ponceau S staining is reversible and will not interfere with antibody labeling of the membrane.
4. Maximize The Number Of Antibodies You Can Test With One Blot
When testing multiple antibodies using the same lysate, try using a stacking layer with one large trough instead of multiple lanes to maximize the number of strips available for testing.
Once the gel has been transferred to a blot, you can use a razor blade and ruler to cut the blot into strips of equal widths. In most cases, twice as many strips can be cut from one large trough blot than can be cut from a blot with multiple lanes. The strips can then be incubated in a tray with narrow wells.
5. A Protein’s Molecular Weight Will Affect Its Transfer Time
Proteins of different molecular weights transfer from gel to membrane at different speeds and efficiencies.
Proteins with larger molecular weights will take longer to transfer than proteins with lower molecular weights. Try staining your gel with coomassie after the transfer to confirm that the proteins of interest have migrated out of the gel. Remember to also consider protein loss across the membrane. By adding a second membrane behind the first, you can see to what extent the proteins have blown through. Process both membranes with your antibody. Use data from such experiments to optimize transfer time for the specific protein of interest.
6. Try BSA for Blocking When Using Phospho-Tyrosine Antibodies
When working with phospho-tyrosine antibodies, try blocking and incubating with 3% and 1% BSA respectively instead of milk.
Milk often contains high concentration of phospho-tyrosine and thus can give an abnormally high background if used as a blocking agent with phospho-tyrosine antibodies.
7. Find Your Optimal Dilution Using A Dilution Curve
Optimal primary antibody dilutions should be determined experimentally using a dilution curve.
A dilution that is too low will give a signal that is oversaturated. A dilution that is too high will give a signal below the detection threshold. Finding the “Goldilocks” zone where the signal is detectable, but not saturated is the goal.
Experimental protocols and imaging systems vary from lab to lab. Trying a dilution curve with the primary antibody allows you to determine the best dilution to use in your system.
8. Store Your Antibody In Aliquots
Antibodies are sensitive and must be handled and stored with care to ensure they remain active and efficient in binding their target proteins.
If you don’t plan on using all of the antibody right away, freeze it in aliquots (volume dependent on application) at -20° C or lower when they are not being used and keep your “working aliquot” stored at 4° C.
Aliquots from antibodies formulated with 50% glycerol in their buffer can be repeatedly taken directly from the stock vial stored at -20° C without freeze/thawing due to the glycerol content.
9. Dilution into working solutions should only be done right before your experiment.
There are two major concerns: antibody stability decreases with increased dilution, and a working solution can encourage microbial growth.
The stability of antibodies, like all proteins, is reduced as dilution is increased due to shearing. Bovine serum albumin is added to many purified antibody solutions to protect the antibody from dilution effects. Microbe growth and contamination in the working solution is much more problematic after transfer from -200C storage to refrigerator storage at 40C and even more so at room temperature. Add antimicrobial agents to a working solution if storage in this form is unavoidable.
10. Combat lysate goop with the right lysis buffer, the right temperature, and sonication.
Preheated, ionic lysis buffer is your best bet for complete lysis.
Using a non-ionic detergent like RIPA buffer won't solubilize membrane and transmembrane proteins. Choose the ionic 1% SDS.
Look after lysing: Look at your plate after lysing and collecting the sample. If cells are still present, lyse again with hot 1% SDS lysis buffer (95˚C).
If there is still a little goop, try sonicating the sample in 5 second pulses at a low frequency.
Finally, heat fix the lysate samples at 95˚C for 5 minutes, and you should be set for Westerns!
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What makes us different?
- If we attempt to make an antibody and it doesn’t work, we throw it away. We never sell an antibody that we can’t get to work in our hands.
- Our commitment to strict validation standards ensures a clean signal in endogenous protein and verifies phosphospecificity for relevant products.
- We are dedicated to reproducibility. Look for our “Pooled Serum” icon. Each antibody with this icon is purified from its own pool of serum to ensure lot-to-lot consistency.
- We guarantee our antibodies. If you can’t get the antibody to work in your application, we will provide a 100% refund.
- When making a custom antibody, we provide personalized customer support from start to finish, from peptide design, to purification, to characterization.