#western blot

[incu-bussy]

GLOW UP

[nemo-duspy-writes]

A small funny tidbit from the realm of Biology/Chemistry:

In 1975, the scientist Edwin Southern developed a method to separate DNA fragments in a gel, then transfer it to a membrane, which makes it possible to label it according to their sequence. The method was named 'Southern blot' to honour its creator.

Later, similar methods were made for RNA and Protein. And these were aptly named 'Northern blot' and 'Western blot'

And I find that beautiful.

[abioegineerinneuroscience]

[chaotic-neutral-medstudent]

actual footage of my summer research project

[biomedicool]

Western Blot

A western blot can detect specific protein molecules from a mixture of proteins.  They can also be used to evaluate the size of a protein of interest, and to measure the amount of protein expression. 

Gel electrophoresis

The proteins of the sample are separated by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. 

Samples are often boiled first to denature the proteins -  prevents proteases (enzymes that break down proteins) from degrading samples.

Most commonly, this uses polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis).

Proteins are covered in the negatively charged SDS - becoming anionic - and migrate towards the positively charged (higher voltage) anode through the acrylamide mesh of the gel. 

Smaller proteins migrate faster through this mesh, and the proteins are thus separated according to size.

The different rates of advancement (different electrophoretic mobilities) separate into bands within each lane. 

The concentration of acrylamide determines the resolution of the gel.

One lane is a marker/ladder - a mixture of proteins of known molecular weights, to act as a control and compare results to so as to estimate the sample’s molecular weight.

Transfer

To make the proteins accessible to antibody detection, they are moved onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). 

Electroblotting uses an electric current to pull the negatively charged proteins from within the gel onto the membrane while maintaining the organization they had within the gel. 

Blocking

Blocking of non-specific binding prevents interactions between the membrane and the antibody used for detection of the target protein. 

Membrane is placed in a dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or non-fat dry milk in tris-buffered saline (TBS) or I-Block, with a minute percentage (0.1%) of detergent 

The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. 

When the antibody is added, it cannot bind to the membrane, and therefore the only available binding site is the specific target protein. 

This reduces background - clearer results, and eliminates false positives.

Primary antibody

A solution of primary antibody (diluted in either PBS or TBST wash buffer) is incubated with the membrane under gentle agitation. 

This binds to the target protein.

The membrane is washed several times in wash buffer to remove unbound primary antibody,

Secondary antibody

Membrane is exposed to another antibody 

Which recognises and binds to the species-specific portion of the primary antibody (eg anti-mouse secondary antibody will bind to almost any mouse-sourced primary antibody).

Secondary antibody is commonly linked to biotin or a reporter enzyme 

Several secondary antibodies will bind to one primary antibody and enhance the signal.

Western blot binding

As with most immuno tests, an enzyme can be attached to the secondary antibody that will produce a coloured reaction product - visible on the membrane so that the test can be read. 

Another method uses a near-infrared (NIR) fluorophore-linked antibody - light produced from the excitation of a fluorescent dye is static, making fluorescent detection more precise and accurate 

A radioactive label could also be coupled to the secondary antibody.

[queenofthebench]

Is it weird that I miss the smell of western blot transfer buffer?

When you take the lid off of a steamin’ hawt transfer and your nose is tickled by the clean smell of dat SDS... *sigh* =_=

[perfectlytinyworkspace]

protocol: western blotting

sample prep and running protein gel

1. Take concentrations from BCA protein assay and diluted to 100-fold (calculated). Total 40ug volume from measured ug/ul concentration. 

2. Calculate lysis buffer amount needed to reach 24 ul/sample x 2.2 for enough to duplicate and 0.2 extra for loading. 

3. Pipet lysed samples to mix before adding volumes from (1) to volumes from (2) and mixing. 

4. Add 10% 2-mercaptoethanol to blue loading dye 7.5.19: 20 -> 200 uL) and vortex to mix. Add 2.2x 8ul/sample to each tube from (3) and pipet to mix. 

5. Vortex gently before incubating @95°C for 5 minutes. Vortex 3s and put on ice. Centrifuge at max rpm for 2 minutes at 4°C. Vortex gently on 1/2 speed.

6. mix 75 uL lysis buffer and 25uL loading buffer for blank practice/addition to marker. No incubation necessary.

7. set up WB gel for loading. Fill to line and inside gel space with running buffer (diluted). (Dilution: 100mL 10x: 1000mL ddH2O). Remember to sub in a plastic plate if you only have 1 gel to run!

8. using butterfly tips, load max 40uL into each well. For gel facing away, R-> load. For gel close to you, L-> R load. 

9. load marker "protein dual core" from -20°C fridge into wells flanking and dissecting gel (2.5uL, 10uL, 10uL). Weigh down marker lanes with 20-25uL soln from (6). 

10. Add H2O and LB/2-mercaptoethanol to extra lanes to prevent marker running sideways. If needed, add more RB while running w/cap on for 2hours (120 min) at 80V

gel-to-membrane transfer process

1. make transfer buffer - 2 gel = 1L. 800 mL H2O, 200mL methanol, 5.8g Tris, 2.9g glycine. Stir w/ teflon and place in ice. 

2. >>transfer order <<i. pour TB in large, flat tray. soak 5 sponges in soln + 4 papers, nitrocellulose membrane.ii. membrane with gel attached will go face down in TB. Start with "u" then flip to "n"iii. [bottom to top] 3 sponge. 2 papers. gel. membrane. 2 papers. 2 sponge. membrane goes to ANODE. <</transfer order>>

3. pry gel out from between glasses to place on membrane, via green rectangle cut below last marker line and above @ gel lanes to remove excess gel. Remove gel lanes at top and excess at bottom. 

4. remove air bubbles w/ TB pour onto gel. Lay paper gently on top and then flip and add membrane. 

5. "make" the sandwich within the white plastic holder. drip each layer with TB using a sponge, until everything is secure and TB overflows. Run transfer 30V for 3-5 hours. Flip on palm, lay down. 

antibody application and binding 

1. rinse with wash buffer and place in plastic compartment with 10-15 mL blocking buffer (2.5g BSA + 50mL wash buffer, shake 20+ minutes before use) per cut. Shake 30min-1hr to reduce noise. 

2. wrap in plastic and close off edges to prevent membrane drying. Cut into sections for antibody binding and snip corners to distinguish between each membrane portion.

3. wash with WB. Add primary antibodies from cold room into each well of a plastic WB container. Place membrane sections into correct antibody lanes. Slowly shake overnight in cold room.

4. wash new container with WB and transfer membranes. Place primary antibodies back in original tubes in cold room. Wash in 8mL WB per well, 10 minutes, 3 times, on shaker at room temperature

5. add 20000x dilution of secondary antibody from protein imaging kit in -20° fridge to prepared 6mL BSA per membrane piece (i.e., 1.25uL rabbit antibody for 25mL BSA) and gently shake on rocker at RT for 1 hr

6. perform another 3 • 10 minutes wash with WB, 6-8mL per well

membrane image capture and b-actin stripping

1. reserve imaging machines in CORE. Bring basket, p1000 pipet, antibody substrate on ice, and membranes with antibody/BSA binding to 9th floor CORE imager 

2. clean imaging screen with ddH2O and immerse each membrane in 250uL antibody substrate on separate dish for 1-2 minutes before placing on screen and imaging. Save files onto USB drive and place membranes back in WB

3. make stripping buffer (recipe in comments). Wash membranes with WB, drain, and place 7.5mL stripping buffer to membranes that contain b-actin/other control bands to remove experimental protein-specific antibody binding

4. block with BSA for 0.5 hr and wash with WB before adding b-actin antibodies from cold room and incubating 1.5+ hours at RT or overnight at 4°C.

5. repeat steps (3 from previous section) - (1 from this section) with b-actin or other control band, to obtain control WB images. Assess control results to ensure consistent protein concentrations and procedures.

6. clean all equipment used with ddH2O and place antibodies, BSA, substrates and kits in cold room or fridge.

[wwalkingdisasterss]

So fucking happy to be done with this