Blocking membrane

1

• Fill 2 50 ml tubes with 5 ml blocking buffer.
• Pick up the membrane (gloves!), roll it up and put the membrane in the tube. Ask the assistants for help.

2

• Put the tubes on the roller bench.
• Block at room temperature for 1 hour.

Now we have the blocking buffer, we need to transfer the membrane in to a 50 ml tube.

What is the correct way to place the PVDF membrane into the tube for blocking?

• Protein-side outward
  Incorrect. There is enough space for the blocking buffer to rich between the the membrane and tube wall to properly cover the whole membrane.
• Protein-side inward
  Very good! The protein side must face inward in order for the blocking buffer to properly cover the membrane.
• The orientation does not matter
  Incorrect. The orientation does matter.

Before we can block our membrane, we need to prepare blocking buffer.

We will make 50 ml of blocking buffer. Calculate how many grams of non-fat dry milk we need to add to TBS-T to make a 5% solution.

We need ____ g of non-fat dry milk

• Of which volume do you need 5%?
• How do you write 5% in decimals?
• Use the formula: (volume/100)*5 ore volume*0,05

Correct answer: 2.5
Very good! 5% of 50 ml is 2,5 g.

De blokbuffer bevat melkpoeder. Waarvoor dient dit?

1. De eiwitten in melk bedekken het membraanoppervlak zodat in de volgende stap het antilichaam niet bindt aan het membraanoppervlak maar alleen aan de eiwitten van interesse.
   Juist! De eiwitten in melk bedekken het membraanoppervlak en zorgen ervoor dat het antilichaam straks uitsluitend bindt aan de eiwitten van interesse. De achtergrondruis wordt daardoor geminimaliseerd.
2. De eiwitten in melk binden uitsluitend aan de eiwitten van interesse zodat deze straks zichtbaar worden en de achtergrond ruis wordt geminimaliseerd.
   Ga nog eens na waaraan de eiwitten in melk binden.

Blocking your membrane is a very important step of Western blotting. But what happens during this step? Wich of the following statements is/are true?

• There are multiple answers correct.
• What do we use as blocking buffer?
• 5% non-fat dry milk /TBS-T contains a lot of proteins. What could be the use of that?

1. Blocking buffer prevents antibodies from binding to the membrane nonspecifically.
   Correct = yes
2. Blocking buffer is a dye that colors all the protein bands.
   Correct = yes
3. Blocking buffer is a solution of irrelevant proteins.
   Correct = yes
4. Blocking buffer activates the membrane so the antybodies can attache.
   Correct = no
5. Blocking buffer passively absorbs to all remaining binding surfaces of the mambrane.
   Correct = no