Phospho-PDGF-β receptor antibody (Tyr751) (2023)

Protocolo de Western Blotting

For Western blots, incubate the membrane with primary antibody diluted in 5% w/v BSA, 1X TBS, 0.1% Tween^®20 to 4°C with gentle stirring overnight.

OBSERVATION: See primary antibody product website for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for western blotting are now included in one convenient kit:#12957Solution kit for western blotting applications

OBSERVATION: Prepare solutions with reverse osmosis deionized water (RODI) or equivalent.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS: add 50 mL of 20X PBS to 950 mL of dH₂Ai mistura
2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1L of 1X TBS: add 100mL of 10X to 900mL of dH₂Ai mistura
3. 1X SDS-Probenpuffer: Blue Cargo Pack (#7722) or red charging pack (#7723) Prepare a fresh 3X reduction loading buffer by adding 1/10 volume of 30X DTT to 1 volume of 3X SDS loading buffer. Dilute 1X with dH₂Ö.
4. 10x Tris-Glicina SDS Laufpuffer: (#4050) Preparation of 1 L 1X Running Buffer: Add 100 mL 10X Running Buffer to 900 mL dH₂Ai mistura
5. 10X Tris-Glicina-Transferpuffer: (#12539) To prepare 1 L of 1x Transfer Buffer: add 100 mL of 10x Transfer Buffer to 200 mL of methanol + 700 mL of dH₂Ai mistura
6. 10X Tris Buffered Saline with Tween^®20 (TBST): (#9997) Preparation of 1L 1X TBST: Add 100mL 10X TBST to 900mL dH₂Ai mistura
7. Fat-free powdered milk: (#9999).
8. Sperrpuffer: 1X TBST with 5% w/v skimmed milk powder; to 150ml, add 7.5g skimmed milk powder to 150ml 1X TBST and mix well.
9. wash buffer: (#9997) 1X TBS.
10. Rinderserum albumin (BSA): (#9998).
11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; to 20mL add 1.0g BSA to 20mL 1X TBST and mix well.
12. Biotinylated Protein Ladder Detection Package: (#7727).
13. Blue pre-stained protein marker, broad range (11-250 kDa): (#59329).
14. Membrane and absorbent paper: (#12369) This protocol has been optimized for nitrocellulose membranes. A pore size of 0.2 µm is generally recommended.
15. HRP-conjugated secondary antibody: Anti-rabbit IgG, HRP-coupled antibody (#7074).
16. detection reagent: SignalFire™ ECL-Reagenz (#6883).

B. Protein Blotting

A general protocol for sample preparation.

1. Treat cells by adding fresh media with regulator for the desired time.
2. aspirating media from cultures; wash cells with 1X PBS; aspire.
3. Lyse the cells by adding 1X SDS sample buffer (100 µL per well of a 6-well plate or 500 µL for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. stay on ice
4. Sonicate for 10-15 seconds to complete cell lysis and cut DNA (to reduce sample stickiness).
5. Heat a 20 µL sample at 95–100 °C for 5 min; cool on ice.
6. Microcentrifuge for 5 min.

7. Carregue 20 µl no gel SDS-PAGE (10 cm x 10 cm).

   OBSERVATION: loading of pre-stained molecular weight markers (#59329, 10 µl/lane) to verify electroblotting and the biotinylated protein ladder (#7727, 10 µl/lane) recommended for molecular weight determination.

8. Electroblot to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

OBSERVATION: The volumes are for 10cm x 10cm (100cm²) the membrane; Adjust volumes appropriately for different membrane sizes.

I. Membrane Blocking

1. (Optional) After transfer, wash the nitrocellulose membrane with 25 mL of TBS for 5 min at room temperature.
2. Incubate the membrane in 25 ml of blocking buffer for 1 hour at room temperature.
3. Wash three times for 5 min each with 15 mL of TBST.

II. Primary antibody incubation

1. Incubate the membrane and primary antibody (using the appropriate diluent and the diluent recommended on the product website) in 10 mL of Primary Antibody Dilution Buffer with gentle shaking overnight at 4°C.
2. Wash three times for 5 min each with 15 mL of TBST.
3. Membrane with Anti-Kaninchen-IgG, antibody linked to HRP (#7074to 1:2000) and anti-biotin, antibody linked to HRP (#7075at 1:1000-1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hour at room temperature.
4. Wash three times for 5 min each with 15 mL of TBST.
5. Proceed with detection (Section D).

D. Protein Detection

Instructions for use:

1. Wash the membrane-bound HRP (antibody conjugate) three times in TBST for 5 minutes.
2. Prepare 1X Reagente SignalFire™ ECL (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (for example, to 10mL add 5mL Reagent A and 5mL Reagent B). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated skin contact.

Flow cytometry, methanol permeabilization protocol for rabbit antibodies

A. Solutions and Reagents

All reagents needed for this protocol can be efficiently purchased together in our intracellular flow cytometry (methanol) kit.#13593, or individually under the catalog numbers listed below.

OBSERVATION: Prepare solutions with reverse osmosis deionized water (RODI) or equivalent.

1. 1X Phosphate Buffered Saline Solution (PBS): To prepare 1 L of 1x PBS: add 100 mL of 10x PBS (#12528) to 900 ml of water.
2. 4% Formaldehyde, ohne Methanol (#47746)
3. 100% Methanol (#13604): Refrigerate before using
4. Antibody Dilution Buffer: Purchase ready-to-use flow cytometry antibody dilution buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g bovine serum albumin (BSA) (#9998) in 100 mL of 1X PBS. Store at 4°C.
5. Recommended secondary anti-rabbit antibodies::
   • Anti-Kaninchen-IgG (H+L), F(ab')2-Fragmento (Alexa Fluor^®488 conjugated)#4412
   • Anti-Kaninchen-IgG (H+L), F(ab')2-Fragmento (Alexa Fluor^®594 conjugated)#8889
   • Anti-Kaninchen-IgG (H+L), F(ab')2-Fragmento (Alexa Fluor^®647 conjugated)#4414
   • Anti-Rabbit-IgG (H+L), F(ab')2-Fragment (PE-Conjugate)#79408

OBSERVATION: If you include fluorescent cell dyes in your experiment (including viability dyes, DNA dyes, etc.), see the dye product page for the recommended protocol. Visitwww.cellsignal.com for a complete listof cellular dyes validated for use in flow cytometry.

B. Fixation

OBSERVATION: Cells or adherent tissues must be dissociated and placed in a single cell suspension prior to fixation.

OBSERVATION: Optimal centrifugation conditions vary depending on cell type and reagent volume. Generally, 150-300 g for 1-5 minutes is enough to pellet the cells.

OBSERVATION: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

OBSERVATION: Antibodies directed to CD markers or other extracellular proteins can be added prior to fixation if the epitope is destroyed by formaldehyde and/or methanol. Antibodies remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and therefore should not be added prior to permeabilization. Do a little experiment if you're not sure.

1. Remove cells from pellet by centrifugation and supernatant.
2. Resuspend cells in approximately 100 µL of 4% formaldehyde per 1 million cells. Mix well to dissociate the granules and avoid cross-linking of individual cells.
3. Fix for 15 minutes at room temperature (20-25°C).
4. Wash with excess 1X PBS by centrifugation. Discard the supernatant in appropriate waste containers. Resuspend cells in 0.5-1mL 1X PBS. Proceed to the Permeabilization step.
   1. Alternatively, cells can be stored overnight at 4°C in 1X PBS.

C. Permeabilization

1. Permeabilise the cells by slowly adding ice-cold 100% methanol to the precooled cells, while gently rotating, to a final concentration of 90% methanol.
2. Permeabilize no gel by hair for less than 10 min.
3. Proceed with immunostaining (section D) or store cells at -20 °C in 90% methanol.

D. Immunostaining

OBSERVATION: Count cells using a hemocytometer or an alternative method.

1. Aliquot the desired number of cells into tubes or wells. (Usually 5x10⁵para 1x10⁶cells per assay.)
2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard the supernatant in appropriate waste containers. Repeat if necessary.
3. Resuspend cells in 100 µL of diluted primary antibody prepared in Antibody Dilution Buffer at the recommended dilution or as determined by titration.
4. Incubate 1 hour at room temperature.
5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard the supernatant. Repeat.
6. Resuspend cells in 100 µL of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
7. Incubate 30 min at room temperature. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard the supernatant. Repeat.
9. Resuspend cells in 200-500 µl 1X PBS and analyze on a flow cytometer.