So I'm new to lab research and learning Western Blots these days. I ordered my secondary antibody and it arrived in a powdered form. Here is the link:
https://www.novusbio.com/products/igg-h-l-antibody_nbp1-75130#protocols-faqs
How to work with this powder to make a solution? It says on the datasheet to dissolve the contents of the bottle into 1.1 ml of water, how to work of that? I previously used antibody from a colleague, it was a liquid form and I put 1 ul on 1 ml to make 1:1000 concentration. How to do that with this powder?
I should say that I have never been involved in lab research before, I'm weak in chemistry of dilutions and still trying to figure it out well. I know the basics and with some heavy calculations my first three weeks on the lab were okay, but I definitely need to relearn the dilutions and concentrations and so on, so any recommendations will be greatly appreciated.
You should be able to have the instructions for it. For example, the one that I work with for a teaching lab says to dissolve the solid in an X amount of ultrapure water PBS prepared solution.
According with your instructions I would spin down the content for brief seconds to make sure that you don't loose powder when you open it, that add the amount of water (ultra pure) and make sure that after you prepare it you split it in several aliquots so you dont have to freeze/thaw several times. Make sure when you run your western blot you have a positive control to check if antibodies are working well
Hi, Ahmad El-Hamamy. I would like to add a few and specific points to your case:
1-Wear a pair of gloves, clean your bench with 70% ETOH, and label about 100 sterilized Epindorff of 600 ul capacity, these will be used to aliquot your antibody.
2- Ask your lab manager where is the laboratory-grade glycerol bottle, you will need 1.1 ml from it later after you reconstitute your antibody.
3- Take your antibody vial out of the refrigerator, keep for 10-15 min at RT for equilibration. spin down briefly at the centrifuge, full power 10-15k rpm for 5-10 secs is fine, the aim is to bring all powder down, don't forget to balance by another tube ask your lab manager how to balance in this in a centrifuge.
4- Open the tube by removing the metal cap and use the P1000 pipette add 550 ul deionized water to the tube twice, not distilled, the one you get from the MilliQ machine or the commercial one used for molecular biology, if not sure ask your lab manager. You can keep the rubber cap and use an insulin syringe instead of the P1000. Allow to stand for 30 minutes on the bench to dissolve.
5- Transfer the contents of your tube to a 15 ml sterile falcon tube and add 550 ul glycerol twice, mix several times by pipetting up and down. now you have 2.2 ml from your antibody at 2x dilution.
5- Use your p20 to aliquot your antibody into the pre-labeled tubes 20 ul each, once you finished, snap freeze all tubes in dry ice, if not sure ask your lab manager.
6- Put all tubes in a plastic bag or inside a dark cart box and store at your -20C space.
Good luck bro.
Dr. Ahmed Abdellatif
I finally understand it thanks a lot, will do this on Monday when I return to the lab, appreciate it.
One last question about this, sorry. What does 2x exactly mean? I still have trouble generally with concentrations and dilutions, I will do my best to get better at it this weekend. For example, how to make a secondary antibody solution in a 1:1000 concentration from these 2x stocks?
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