Biotinylated Antibodies (2023)

For determination of protein concentration in cell culture

Discard the medium from a 10 cm plate of adherent cells and wash the plate briefly with phosphate-buffered saline (PBS).
Aspirate excess PBS.
Add 1 ml of boiling lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, 10 mM Tris pH 7.4).
Scrape the cells from the plate, transfer to a microcentrifuge tube and boil for 5 min in a boiling water bath. To reduce viscosity, the sample can be sonicated briefly or passed through a 26-gauge needle several times.
Centrifuge the sample for 5 min to pellet the insoluble material and collect the supernatant, which contains the cell lysate.
Dilute an aliquot of the lysate sample at least 10-fold for BCA (Pierce) protein concentration determination (SDS concentration must be below 0.1% to avoid interfering with the colorimetric reading).

For cell culture protein gel electrophoresis (without determination of protein concentration)

Discard the medium from a 10 cm plate of adherent cells and wash the plate briefly with 1X phosphate buffered saline (PBS).
Aspirate excess PBS.
Add 1 ml of boiling 2X concentrated electrophoresis sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% β-mercaptoethanol).
Scrape the cells from the dish, transfer to a microcentrifuge tube and boil for another 5 minutes. To reduce viscosity, the sample can be briefly sonicated or passed several times through a 26 needle. Centrifuge the sample for 10 min to precipitate the insoluble material and collect the supernatant (lysate).
The lysate sample is now ready to load onto the gel.

For determination of whole tissue protein concentration

Rapidly homogenize each 0.25 g of tissue in 3.5 ml of boiling lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, 10 mM Tris pH 7.4).
Microwave for 10–15 seconds.
Centrifuge the homogenate (16,000 x g, 15°C) for 5 min to pellet insoluble material, then discard the pellet.
Dilute an aliquot of the tissue lysate sample at least 10-fold for determination of BCA protein concentration (Pierce).

POLYACRYLAMIDE GEL ELECTRIFICATION

Guidelines for selecting the gel percentage to use for certain molecular weight proteins (based on a 37:1 acrylamide:bisacrylamide ratio)

4-5% gel: > 250 kDa

7.5% gels: 250-120 kDa

10% Gel: 120-40 kDa

13% gels: 40-15 kDa

Gel electrophoresis

If the cell lysate is not already in electrophoresis sample buffer, add an equal volume of 2X sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, β-1.8% mercaptoethanol) to all samples and boil for 3-5 minutes.
Apply 5-20 µg of total protein from cell or tissue lysate to each well of a 0.75-1.0 mm thick gel. For thicker gel (1.5 mm thickness), apply up to 25-40 µg to each well. Consult the antibody data sheet for the appropriate positive control cell lysate.
Electrophoresis until the bromophenol blue in the samples reaches the bottom of the gel. Turn off the power supply. Keep the buffered gels running until ready for transport.

PROTEIN SCAFFOLDING

liquid transport

Transport buffer: 25 mM Tris, 190 mM glycine, 20% MeOH, pH adjusted to 8.0.

Note: Because additional negative charges are required to reach 1 Amp in a liquid transport system, adjust the pH of the transport buffer to approximately pH 8.0 using NaOH.

To transfer proteins smaller than 20 kDa, transfer proteins from the gel to the PVDF (polyvinylidene difluoride) membrane with a constant current of 1 Amp for 45 min or equivalent (250 mAmp for 3 h or 500 mAmp for 90 min) in transfer buffer.
For transfer of proteins smaller than 120 kDa, transfer proteins from the gel to the PVDF membrane at a constant current of 1 Amp for 1 hour or equivalent (250 mAmp for 4 hours or 500 mAmp for 2 hours) in transfer buffer.
For proteins larger than 120 kDa, transfer to PVDF membrane at a constant current of 1 Amp for 90 min or equivalent (250 mAmp for 6 h or 500 mAmp for 3 h) in transfer buffer + SDS.
For proteins larger than 250 kDa, transfer to PVDF membrane at a constant current of 1 Amp for 1 hour 45 minutes or equivalent (500 mAmp for 3.5 hours) in transfer buffer + SDS.

* To ensure complete transfer of high molecular weight proteins (as in 3 and 4 above), 0.05% SDS can be added to the transfer buffer.

Semi-dry transport

For protein transfer from 10% or 13% gels to PVDF membranes, semi-dry transfer can also be used. Transfer the proteins to the PVDF membrane at 1.2 mAmp/cm2 for 1 h 45 min in transfer buffer.

Optional

If no blots are used for colorimetric detection, visualize transferred proteins by staining the membrane for 15 min with India ink (Higgins black ink, Eberhard Faber) diluted 1:1000 in wash buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20). Wash off excess stain with wash buffer before blotting.

Ponceau S can be used if the blot is to be used for colorimetric tests, as it is a reversible dye. Stain with 0.1% Ponceau S for 5 min and then visualize the protein bands. To remove, rinse with distilled water and then soak in 0.1M NaOH aqueous solution until the bands disappear (10 to 30 seconds), then rinse the membrane with distilled water before blocking.

block

FOR ALL ANTIBODIES EXCEPT PHOSPHOTYROSINE

Remove the blot from the transfer device or staining tray and immediately place in blocking buffer (5% skimmed milk powder, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20).
Incubate the blot for 30 min at 37°C, 1 h at room temperature, or overnight at 4°C.

FOR PHOSPHOTYROSINE ANTIBODIES

Remove the stain from the transfer device or staining tray and immediately place in blocking buffer (1% BSA, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20).
Incubate the blot for 30 min at 37°C, 1 h at room temperature, or overnight at 4°C.

Incubation with biotinylated primary antibody

primary antibody

Dilute the antibody in the corresponding blocking buffer.
Decant transfer blocking buffer, add antibody solution, and incubate with shaking for 30 min at 37°C, one hour at room temperature, or overnight at 4°C.

Enzyme conjugate incubation

NOTE: The inclusion of sodium azide should be avoided in all steps using HRPO (Horseradish Peroxidase) conjugates.

Decant the primary antibody solution, add wash buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20) and wash for 30 min with shaking, changing wash buffer every 3-5 min.
Dilute the strepavidin:HRPO conjugate to the supplier's recommended dilution in blocking buffer (3% BSA, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20).
Decant the wash buffer, add the diluted enzyme conjugate and incubate with shaking for 30 minutes at 37°C or one hour at room temperature.

SUBSTRATE INCUBATION

Decant the secondary antibody solution, add wash buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20) and wash for 30 min with shaking, changing the wash buffer every 3-5 min.
Decant the wash buffer and place the blot in a plastic bag or clean tray containing chemiluminescence working solution (0.125 ml/cm2). Swirl the bag or tray to allow the solution to cover the membrane surface for 1 to 5 minutes.
Remove the blot from the bag or tray and place it between two pieces of acetate writing transparency. Spread over the covered stain to remove air bubbles and excess substrate.

EXPOSE TO X-RAY FILM OR ANY SENSITIVE SCREEN. AN INITIAL EXPOSURE OF 10-60 SECONDS IS RECOMMENDED FOR FILM.

PROBLEMS SOLVED

Weak signal or protein of interest is expressed at low levels in the sample:

Try using the recommended positive control solution.
Increase primary antibody concentration.
Increase the amount of total protein loaded on the SDS gel.

Insufficient protein transport

Blot with India ink or Ponceau red to ensure that the proteins have been transferred to the membrane.
Be sure to use the appropriate transport conditions for the protein of interest. For high molecular weight proteins, use SDS in the transfer buffer.

The substrate is inactive

Use fresh substrate. Check the secondary antibody and HRP-conjugated substrate to make sure they are working properly.

high bottom

Probably due to insufficient blocking. use 5% milk, block more and increase wash and wash time.

The target protein was digested:

Use SDS detergent in your sample buffer and include protease inhibitors.