Western blot | Re-use a primary antibody

By -

Why reuse primary antibody?

Western blotting is one of the most common method for detecting and semi-quantifying specific proteins. The cost of running a Western is predominantly driven by detection antibodies, which are used to probe membrane-bound target protein(s). Although some antibodies can be produced cheaply in-house, most antibodies must be commercially ordered. Reusing antibodies for subsequent Westerns can save your lab a considerable amount of money. So money is nothing but everything. In conclusion economic research practises is budget friendly and reuse of primary antibody is one of them. 
reduce primary antibody cost


When reuse is problematic?

  • The most standard phospho-antibody protocols involve blocking with 5% milk in PBS or TBS (0.1% Tween 20), followed by a couple washing steps with PBS or TBS to remove the excess milk. If you dilute your antibody in a milk solution, storage and re-use will be extremely problematic, because the milk will "go bad" and fall out of solution, similar to milk at home. 
  • BSA is ideal for your needs. Dilute your primary antibody in 5% BSA in PBS or TBA (0.1% Tween 20) [1]. 
  • Generally milk is not used for blocking if its a phospho antibody. You can store used antibody in 4C for upto 2 weeks [2].

When and how we reuse primary antibody?

  • Use 1% non-fat milk for antibody preparation. If you want to use further then give pri Ab treatment at 4 deg for overnight or at least 4 h. Later, store at -20 deg. You can use easily 2-3 times. It works for most of the antibodies [3]. 
  • Both myself and my lab mates dilute our primary antibodies in 5% BSA in TBST with Sodium Azide stored at 4C for months to over a year and typically use them several times a month to weekly with great results [4].
  • Hii, Dilute primary antibodies in TBST with 5% BSA. Usage of milk can create non-specific background, because casein itself is a phosphoprotein. Further, milk in TBST can not be stored for long at 4 degree C. you can store your primary antibody at 4 degree C for 2-3 weeks and can reuse 2-4 times efficiently [5]. 
  • You may use milk but BSA is better if you plan to store your diluted antibody longer. Use sodium azide (0.05%). Even with azide, milk in your dilution buffer will get spoiled thereby affecting long term storage. Have you ever tried diluting your phospho antibodies in just plain PBS without any blocking agent? You are probably doing the blocking step prior to your antibody step right?[6].  
  • It all depends on how strong the signal you get with the previously used antibodies and how stable they are. Also, it depends on the working titer of the antibodies and how much of the antibodies were consumed during your first use of them. My advice to everybody is to standardize that by rechecking the reactivity of previously used antibodies on strips from the same target protein and see if the signal remains as strong or gets weaker upon use [7].
  • We design and produce phospho antibodies in our lab.  We block our antibodies with serine and threonine phospho sites with 5% non-fat milk.  We use BSA for phospho tyrosine antibodies because the phospho tyrosine in milk can produce high background.   It is true that milk goes bad faster than BSA, and BSA will certainly work for all antibodies.  The azide is great as long as it doesn't interfere with your system.  We divide our antibodies into working aliquots, and only keep one aliquot in 4 degrees at a time.  As mentioned in other posts, we add glycerol to 50% and store at -20 to avoid freeze/thaw.  For long term storage, we recommend aliquoting and storing at -80.  We generally don't reuse our antibodies, but here is an idea to reduce the amount of antibody you use when testing multiple antibodies using the same lysate: Try a gel with a stacking layer that has one large trough instead of one that has multiple lanes. Once the gel has been transferred to a blot, you can cut the blot into strips of equal widths. In most cases, twice as many strips can be cut from one large trough than from cutting apart a blot with multiple lanes. The strips are also more narrow and require much less incubation solution [8]. 

Comments

Post a Comment

Popular posts from this blog

Cleaning Validation | Campaign Cleaning Validation (CCV)

By -
Image
When the product demand does not allow for a complete clean up after each batch, a campaign cleaning validation must be conducted.   A back to back cleaning is performed between batches in a campaign, and a complete cleaning at the end of a campaign. Sampling of the equipment is performed after the last batch in a campaign, and all test results must meet predetermined acceptance criteria as per campaign cleaning validation protocol.   Samples for residual active may be taken after back to back cleanings where possible, for information only . Microbiology samples may be taken after back to back cleanings in a campaign to ensure that there is no excessive microbial growth on the equipment.
Read more

Western blot | troubleshooting | samples turn yellow and precipitate

By -
Image
Why western Blot samples turn yellow and precipitate? Sample appears to be acidic. Bromophenol blue in the loading buffer turns to yellow at acidic pH (below 3). Try to adjust the pH by adding NaOH [ 1 ].  Reason for that is proteins may be getting degraded due to heat exposure. One researcher had the same problem and so instead of keeping samples at 95 ° c  he started warming at 60-65 °  C. So anyone can try it out and as [ 1 ] said check the pH as well of the loading dye [ 2] .  Heating a sample also changes pH. An altered pH is not only responsible to turn the sample color yellow, but may also precipitate the proteins. Keeping SDS containing samples in ice is another common mistake to see this precipitation [ 3 ].  Another researcher said that he has got to do more with the total protein concentration in the lysate. He faced the same problem of precipitation upon denaturation at 100 ° c . He added 1/10th volume of 10% SDS and then boiled. Protein did not precipitate. T
Read more

Blending | Effective Sampling During Process Validation | Optimize Sampling Location

By -
Image
Important of sampling during blending: Sampling plays a great role in achieving the accurate results of analysis. Sampling must and procedure must be defined in validation protocols and training should be provided to the concerned staff before the validation activities start. Samples are representative of the whole batch. So it should be handled and weight carefully because segregation may occur during weighing and transpiration. Sample quantity should not be more than the required and whole mass sample should be used in the analysis. Fig: Blending Machine Sampling for blend: Blends are tested for their homogeneity. Homogeneity of the blend is critical for the quality of the final product. The sample should be taken from at least 10 locations in the blender. The sampling location should be selected according to the difficulty of the blending. Ares of poor blending must be covered in sampling. Corners and discharged point must have sampling location.
Read more