Myosin IIa antibodies (2023)

Western blot protocol

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.

USE: Refer to the primary antibody product page for recommended antibody dilution.

A. Solutions and Reagents

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

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

1. 20X phosphate buffered saline (PBS): (#9808) To prepare 1 L of 1X PBS: add 50 mL of 20X PBS to 950 mL of dH₂Hallo Mix.
2. 10X Tris-buffered saline (TBS): (#12498) To prepare 1L of 1X TBS: add 100mL of 10X to 900mL of dH₂Hallo Mix.
3. 1X SDS-Probenpuffer: Blue Load Pack (#7722) or the red cargo package (#7723) Prepare a new 3X Reducer Loading Buffer by adding 1/10 volume 30X DTT to 1 volume 3X SDS Loading Buffer. Dilute 1X with dH₂Ö
4. 10x Tris-Glycin SDS Laufpuffer: (#4050) To prepare 1L of 1X Run Buffer: Add 100mL of 10X Run Buffer to 900mL of dH₂Hallo Mix.
5. 10X Tris-Glycin-Transferpuffer: (#12539) Preparation of 1 L of 1x Transfer Buffer: add 100 mL of 10x Transfer Buffer to 200 mL of methanol + 700 mL of dH₂Hallo Mix.
6. 10X Tris-buffered saline with interpolation^®20 (TBST): (#9997) Preparation of 1L 1X TBST: Add 100mL 10X TBST to 900mL dH₂Hallo Mix.
7. Magermilchpulver: (#9999).
8. Crash-Puffer: 1X TBST with 5% w/v skimmed milk powder; For 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; For 20mL add 1.0g BSA to 20mL 1X TBST and mix well.
12. Biotinylated protein ladder detection package: (#7727).
13. Prestained blue protein marker, large range (11-250 kDa): (#59329).
14. membrane and blotting paper: (#12369) This protocol has been optimized for nitrocellulose membranes. In general, a pore size of 0.2 µm is recommended.
15. HRP conjugated secondary antibody: anti-rabbit IgG, antibody linked to HRP (#7074).
16. detection reagent: Reaktives SignalFire™ ECL (#6883).

B. Proteintransfer

A general protocol for sample preparation.

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

7. Cargue 20 l Gel SDS-PAGE (10 cm x 10 cm).

   USE: loading of prestained molecular weight markers (#59329, 10 µl/lane) to check the electroblotting and the biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights.

8. Electroblot on nitrocellulose membrane (#12369).

C. Membrane blocking and antibody incubations

USE: Volumes are for 10cm x 10cm (100cm²) membrane; Adjust volumes accordingly for different sized membranes.

I. Membranblock

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 diluent as recommended on the product page) 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. Incubate the membrane with anti-rabbit IgG, HRP-linked antibody (#7074to 1:2000) and anti-biotin, antibody linked to HRP (#7075a 1:1000–1:3000) for the detection of biotinylated protein markers in 10 ml of blocking buffer with gentle shaking for 1 hour at room temperature.
4. Wash three times for 5 min each with 15 mL of TBST.
5. Proceed with screening (Section D).

D. Proteinnachweis

Instructions for use:

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

* Avoid repeated skin contact.

Immunofluorescence (immunocytochemistry)

A. Solutions and Reagents

Get high-quality immunofluorescence images with efficient and inexpensive prepackaged reagents in our#12727Immunofluorescence application solution kit

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

1. 20X phosphate buffered saline (PBS):(9808) To prepare 1 L of 1X PBS: add 50 mL of 20X PBS to 950 mL of dH₂Hello Mix. Adjust the pH to 8.0.
2. Formaldehyde: 16%, no methanol, Polysciences, Inc. (Cat# 18814), use new vials and store uncapped at 4°C in the dark, dilute in 1X PBS for use.
3. Crash-Puffer(1X PBS / 5% normal serum / 0.3% Triton™ X-100): To make 10mL, add 0.5mL of normal serum of the same species as the secondary antibody (e.g. normal goat serum (#5425)) y 0,5 ml 20X PBS a 9,0 ml dH₂Mix well. Add 30 µl of Triton™ X-100 while stirring.
4. Antibody Dilution Buffer(1X PBS / 1% BSA / 0.3% Triton X-100): To make 10 mL, add 30 µL of Triton™ X-100 to 10 mL of 1X PBS. Mix well and add 0.1 g BSA (9998), Mix.

5. Recommended fluorochrome-conjugated anti-rabbit secondary antibodies:

   • Rabbit-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® 488 Conjugate) # 4412
   • Rabbit-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® 555 Conjugate) # 4413
   • Kaninchen-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® Conjugate 594) # 8889
   • Kaninchen-Anti-IgG (H+L), F(ab')2-Fragment (Alexa Fluor® Conjugate 647) # 4414
6. Expand^®Gold-Antifade-Reagenz(#9071),Expand^®Gold AntiFade Reagent with DAPI(#8961).

B. Sample preparation: cultured cell lines (IF-IC)

USE: Cells must be cultured, treated, fixed, and stained directly on multiwell plates, chamber slides, or coverslips.

1. Aspirate the liquid and then cover the cells to a depth of 2-3 mm with 4% formaldehyde diluted in 1X PBS.

   USE: Formaldehyde is toxic, use only in a fume hood.

2. Allow cells to sit at room temperature for 15 min.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with immunostaining (section C).

C. Immunostaining

USE: All subsequent incubations, unless otherwise specified, should be performed at room temperature in a light-tight humid box or covered dish/dish to prevent drying and fading of the fluorochrome.

1. Block the sample in blocking buffer for 60 min.
2. During the block, prepare the primary antibody by diluting it as directed on the product page under Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate the sample in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1 to 2 hours at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Coverslips with prolong^®Gold-Anti-Fade-Reagenz (#9071) or extend^®Gold Antifade Reagent with DAPI (#8961).
9. For best results, allow the mounting medium to set overnight at room temperature. Store slides flat at 4°C and protected from light for long-term storage.